LO-CD2a antibody and uses thereof for inhibiting T cell activation and proliferation

ABSTRACT

The present invention relates to a LO-CD2a antibody and methods of using such antibodies or molecules that bind to the same epitope (or a portion thereof) to prevent and inhibit an immune response in human patients, preferably, where the immune response is mediated by the activation and proliferation of T cells or natural killer cells. The administration of an effective amount of the LO-CD2a antibody to a human patient will prevent or inhibit graft rejection, graft versus host disease or autoimmune disease.

This invention relates to an antibody (or fragment or derivativethereof) and preferably, to an antibody (or fragment or derivativethereof) which binds to human lymphocytes. More particularly, thisinvention relates to preventing and/or inhibiting on-going immuneresponses in a patient through the administration of such antibody (orfragment or derivative thereof) to a patient. Preferably, this inventionrelates to preventing or inhibiting T cell activation and proliferationthrough the administration of such antibody or fragment or derivativethereof to a patient.

The prior art has disclosed the possibility of using antibodies to CD2antigen for inhibiting graft rejection. In general, the prior artdiscloses the use of antibodies which bind to CD2 antigens as beingpossibly useful for inhibiting graft rejection, see, OrthoPharmaceutical Corp., U.S. Pat. Nos. 4,364,973; 4,614,720; 4,515,893;4,743,681; and 4,798,806.

Such antibodies have not been known to be useful in inhibiting graftrejection in human patients or in non-human primates. As exemplified inthe following references, J. V. Giorgi, et al., Immunosuppressive Effectand Immunogenicity of OKT11A Monoclonal Antibody in Monkey AllograftRecipients, Transplantation Proceedings Vol. XV No. 1, March 1983, andP. J. Thurlow, et al., A Monoclonal Anti-Pan-T-Cell Antibody,Transplantation, Vol. 36, No. 3, Pg. 293-298.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1

Two color staining of peripheral blood mononuclear cells (PBMC) withbiotinylated LO-CD2a and Leu-5bPE.

For this staining, the following parameters were followed:

PARAMETER: FLI-H/(LOG) FL2-h(LOG) QUAD LOCATION: 17.15,9 TOTAL = 5000GATED = 1290 QUAD EVENTS % GATED % TOTAL X MEAN Y MEAN 1UL 299 23.183.98 11.41 284.69 2UR 831 65.97 17.02 32.70 630.65 3LL 135 10.47 2.704.00 3.31 4LR 5 0.39 0.10 25.11 6.54

FIG. 2

Human PBMC were stained with LO-CD2a-FITC and then a) stained withT11-PE(Coulter antibody to CD2) conjugated with phycoerythrin (PE) or b)Leu-5B-PE (Becton Dickinson antibody to CD2) conjugated to phycoerythrin(PE). In neither case was staining by the second antibody altered bypretreatment with LO-CD2a.

FIGS. 3 a and 3 b

Effects of LO-CD2a on membrane markers. PBMC at 2×10⁶ cells/ml werecultured in the absence (solid lines) or in the presence (broken lines)of LO-CD2a (200 ng/ml). At the times indicated in the figures, cellswere harvested and treated for cytofluorometric analysis. a) and b);PBMC were labeled with anti-CD3 (Leu-4a-FITC), anti-CD4 (T4-RD) mAbs,anti-class II antigens (LO-DRa-FITC) or anti-CD8 (T8-RD) monoclonalantibodies (mAbs). Negative controls for commercial mouse mAbs werealiquots of the same cells stained with FITC or Rhodamine-labeled mouseIgGs. Negative controls for rat mAbs were cells incubated with normalrat serum followed by a FITC-labeled mouse anti-rat mAb (MARK-FITC).Results are expressed as percentage positive cells.

FIG. 4

Effects of LO-CD2a on membrane markers and human blood lymphocyteculture with and without addition of LO-CD2a. Lymphocyte Cultures at1×10⁶ cells ml were labeled with (a) anti-CD2 (Leu-5b-FITC), anti-CD4(T4-RD1) mAb, or anti-CD8 (T8-RD) mAb at times indicated. Negativecontrols for commercial mouse mAbs were aliquots of the same cellsstained with FITC or Rhodamine-labeled mouse IgGs. Negative controls forrat mAbs were cells incubated with normal rat serum followed by aFITC-labeled mouse anti-rat mAb (MARK-FITC). Results are expressed aspercentage of positive cells.

FIGS. 5 a and 5 b

Effects of LO-CD2a and Leu-5b on CD2 expression. Human PBMC wereincubated with a) LO-CD2a (200 ng/ml) or b) Leu-5b (dialyzed againstPBS, diluted 1:2) for the times indicated and a) stained for expressionof CD2 (Leu-5b-FITC and T11-RD1) and for binding of LO-CD2a(Mark-3-FITC) or b) CD2 (LO-CD2a-FITC, T11-RD1) and for binding ofLeu-5b Goat anti-mouse (GAM-FITC).

FIG. 6

Effects of LO-CD2a on MLR. a) inhibition of MLR in mixed lymphocytecultures incubated for 6 days in the presence of increasingconcentrations of LO-CD2a added at time 0. Cultures were harvested atday 6; b) inhibition of MLR in mixed lymphocyte cultures incubated withdifferent concentrations of LO-CD2a added at time 0. Cultures wereharvested at 24 h intervals; c) ³H-Thymidine (³H-T) incorporation (cpm)by mixed lymphocyte cultures in the absence (solid line) or in thepresence (broken line) of LO-CD2a (200 ng/ml); d) inhibition of MLR byLO-CD2a (200 ng/ml) added at different times after the start ofincubation. Cultures were harvested at day 6. All cultures were made intriplicate (1×10⁶ cells of each donor/ml) in a final volume of 200μl/well. ³H-Thymidine was added 8 h before harvesting cultures. Resultsin c) are shown as cpm ×10⁻³ incorporated per well harvested at the timeindicated. Results in a), b) and d) are expressed as percentageinhibition of MLR of triplicate cultures (mean ±S.D.), as compared tocontrol cultures (without LO-CD2a).

FIG. 7

Effects of LO-CD2a on blast cells during MLC. Peripheral bloodmononuclear cells were cultured in mixed lymphocyte cultures with orwithout the addition of 200 ng/ml of LO-CD2a. At the indicated timescells were removed and analyzed by flow cytometry after staining withantibody to CD2 (Leu5b-FITC). Blast cells were gated by forward and sidescatter and the expression of the indicated markers quantified on theblast cells.

FIGS. 8 a and 8 b

Effects of LO-CD2a on resting cells during MLC. Human lymphocytes werecultured with and without the addition of LO-CD2a (200 ng/ml). At theindicated times cells were removed and stained for CD3 (Leu 4a-FITC),CD4 (T4-RD1) CD8 (T8-RD1) or CD25 (LO-TACT-1-FITC). The restinglymphocytes were identified by differential gating for size andgranularity and the results are expressed as the per cent of totalresting lymphocytes staining with the indicated antibody. (FIG. 8 a.)The percentage of resting cells positively stained by Leu-5b in cultureswith and without LO-CD2a is shown in FIG. 8 b.

FIG. 9

Effects of LO-CD2a on mitogen-stimulated lymphocytes. PBMC were culturedfor 96 h in the absence or in the presence of OKT3 (100 ng/ml), Con-A(10 μg/ml) and PHA (1 μg/ml). In parallel cultures, LO-CD2a (200 ng/ml)was added 1 h after mitogens (gray bars) or 1 h before mitogens (blankbars). Checked bars represent cultures performed in the presence ofLO-CD2a alone. Cultures (in triplicate) were pulse-labeled with³H-Thymidine during the last 8 h of incubation.

FIG. 10

Effects of LO-CD2a on mitogen-driven activation of PMBC. PMBCs from twodonors were cultured for 96h in the presence of OKT3 (100 ng/ml), CON-A(10 μg/ml) and PHA (1 μg/ml). In parallel cultures, LO-CD2a (200 ng/ml)was added at Day 0 (0 h) after the initiation of the culture, Day 1 (24h), or Day 2 (48 h). The graph depicts the percentage inhibition byLO-CD2a of the mitogen-induced proliferation in each donor.

FIGS. 11 a and 11 b

Inhibition of NK activity by incubation of the effector and target cells(⁵¹CR labeled K562 cells) in the presence of LO-CD2a. Threeconcentrations of LO-CD2a have been tested: 5 μg/ml, 1 μg/ml, 0.5 μg/ml.Effector cells were peripheral blood lymphocytes of NK activity isexpressed as percent lysis of labeled target cells. Two normal subjectswere tested at 3 E/T ratios: 200/1, 200/1, 50/1.

FIG. 12

Total lymphocytes per μl of peripheral blood of a cynomolgus monkeyreceiving 20 mg/day of LO-CD2a for 10 days (days 0-9).

FIG. 13

PBMC from the cynomolgus monkey receiving LO-CD2a at 20 mg/day for 10days (day 0 to 9) were stained with monoclonal antibodies to CD2(Leu-5b), CD4 (Leu3a), CD8 (Leu 2a), Natural Killer cells (CD8 andCD11b), and B cells (anti-IgM) on the days indicated and analyzed byflow cytometry. Results are presented as the percentage of the totalnumber of staining cells per microliter of blood.

FIG. 14

NK activity of a cynomolgus monkey receiving 20 mg/day of LO-CD2a for 10days (day 0-9). NK activity was assayed on days 11 and 22 and presentedas % lysis at E/T of 25/1, 50/1 and 100/1.

FIG. 15

Serum concentration of LO-CD-2a of a cynomolgus monkey receiving theantibody at 20 mg/day for 10 days (day 0-9). The monoclonal antibody wasmeasured by ELISA as described in the text and expressed in μg/ml.

FIG. 16

Development of IgG antibody to LO-CD2a in a cynomolgus monkey receiving20 mg/day of LO-CD2a for 10 days (days 0-9). The antibody to themonoclonal antibody was measured in serial dilutions of serum drawn onthe indicated days by the sandwich ELISA described in the text and isexpressed as the optical density at 492 nm.

FIGS. 17 a and 17 b

Effect of LO-CD2a on baboon lymphocytes.

a) On the days indicated blood was obtained from the baboon and cellswere stained with the anti-CD2 antibodies T11-RD1 and Leu-Sb-FITC,LO-CD2a and MARK3-FITC, a mouse anti-rat kappa 1b antibody coupled toFITC to detect bound LO-CD2a.

b) Serum samples taken on the indicated days were evaluated for levelsof LO-CD2a by ELISA.

FIGS. 18 a and 18 b

Effect of LO-CD2a on baboon lymphocytes.

On the indicated days blood was taken and the cells stained to detectbound LO-CD2a with MARK3-FITC and MARK2b-8-biotin (a mouse monoclonalanti-rat IgG2b antibody coupled to biotin) detected with PE-coupledstreptavidin).

a) No pretreatment of cells;

b) incubation with 2.5 μg/ml LO-CD2a prior to staining to detect anysites unoccupied by circulating antibody.

FIG. 19

Effect of LO-CD2a on baboon lymphocytes.

On the indicated days blood samples were taken and stained with T4-RD1(CD4); T8-RD1 (CD8) or MARK3-FITC (bound LO-CD2a).

FIG. 20

Leukocytes, lymphocytes and creatinine in patient #1 treated with ATGthen LO-CD2a for allograft rejection.

FIG. 21

Serum levels of LO-CD2a during and following treatment in patient #1.

FIG. 22

Creatinine in patient #2 prior to, during and following treatment withLO-CD2a.

FIG. 23

Leukocyte and lymphocyte counts in patient #2 prior to, during andfollowing treatment with LO-CD2a.

FIG. 24

Serum levels of LO-CD2a in patient #2, drawn just prior to and 2.5 hoursafter each injection.

FIG. 25

Leukocyte count, lymphocyte count and serum creatinine level in patient#3 receiving LO-CD2a for rejection of renal allograft.

FIG. 26

Dual color staining with LO-CD2a and (2) Leu5b, (3) Leu 4(CD3), (4)Leu3a(CD4), (5) Leu2b(CD8) and (6) Leu11 (anti-CD16) a marker for NKcells. LO-CD2a binding was detected with goat anti-rat IG-FITC. Theupper set (1-6) of two color histograms shows the double staining. Thelow set (7-12) shows single staining with each antibody.

FIG. 27

Two color staining of human PBL with a rat isotype control for LO-CD2a(Pharmingen, purified rat IgG2b, kappa) or LO-CD2a and phycoerthyrinconjugated antibodies to CD4 (c,d), Cd8 (e,f), CD16(g,h), CD19(i,j) andCD2 (k,l). LO-CD2a and the isotype control were detected with FITCconjugated affinity purified F(ab′)2 anti-rat immunoglobulin (SouthernBiotechnology). The antibodies to the CD antigens were all phycoerythrinconjugated antibodies obtained from Becton-Dickinson [CD4(Leu3a),CD8(Leu2a), CD16(Leu-11b), CD19(Leu 12) and CD2 (Leu5b)]. In each casestaining with the isotype control is shown in the first histogram andthe LO-CD2a in the second histogram. Histogram a shows the pattern withthe isotype control and b with LO-CD2a.

FIG. 28

Cytofluorograph analysis of the staining of COS cells transfected withwild-type CD2. The left panels show the histograms of staining of a COScell transfected with the control vector, not containing CD2; the rightset of panels staining of a COS cell transiently transfected with avector containing the entire CD2 molecule. In each set the top histogramshows the staining with murine W632 (antibody to Class I, known to beexpressed by COS cells) and 76-2-11 (an isotype control for the murineW632); the middle panel shows staining with Leu5b (anti-CD2 from BectonDickinson) and 76-2-11. an isotype matched control for Leu5b staining,the bottom panel staining with LO-CD2a and a rat isotype matched controlfor LO-CD2a.

FIG. 29

Nucleotide (SEQ ID NO:91) and amino acid (SEQ ID NO:92) sequences of thechimeric LO-CD2a V_(L) chain.

FIG. 30

Nucleotide (SEQ ID NO:93) and amino acid (SEQ ID NO:94) sequences of thechimeric LO-CD2a V_(H) chain.

FIG. 31

Amino acid sequences of the light chain variable region of rat LO-CD2a,(SEQ NO ID:95) human HUM5400 (SEQ ID NO:96), and humanized LO-CD2a (SEQID NO:97).

FIG. 32

Nucleotide (SEQ ID NO:98) and amino acid (SEQ ID NO:99) sequences of thehumanized LO-CD2a variable region.

FIG. 33

Amino acid sequences of the heavy chain variable region of rat LO-CD2a(SEQ ID NO:100), human Amu5-3(SEQ ID NO:102), and humanized LO-CD2a (SEQID NO:101).

FIG. 34

Nucleotide (SEQ ID NO:103) and amino acid (SEQ ID NO:104) sequences ofthe humanized LO-CD2a heavy chain variable region.

FIG. 35

Binding of rat LO-CD2a, humanized LO-CD2a to Jurkat cells.

FIG. 36

Induction of hyporesponsiveness in vitro by rat LO-CD2a, humanizedLO-CD2a, and control rat and human immunoglobulins.

FIGS. 37A, 37B, and 37C

Inhibition of primary MLR by LO-CD2a and response of T-cells culturedwith LO-CD2a in a primary MLR to an antigen in a secondary MLR or athird party stimulator in an MLR.

FIGS. 38A and 38B

Response of T-cells cultured with LO-CD2a in a primary MLR to an antigenin a secondary MLR or to tetanus toxoid in secondary cultures.

FIG. 39

Effect of F(ab′)₂ fragment of LO-CD2a on an MLR.

FIG. 40

Comparison of inhibitory properties of intact LO-CD2a antibody with theF(ab′), fragment of LO-CD2a on the proliferation of PBMC by solubleOKT3.

FIG. 41

Effect of APCs on inhibitory properties of LO-CD2a on proliferationinduced by plate bound OKT3.

FIG. 42

Amino acid sequences of the heavy chain variable region of rat LO-CD2a(SEQ ID NO:100), the humanized antibody MEDI-507 (SEQ ID NO:105), andhuman Amu5-3 (SEQ ID NO:102).

FIG. 43

Plasmid maps of hcmv-Vllys-kr-neo and hcmv-VhLys-gamma1-neo.

FIG. 44

Plasmid maps of pEE6hCMV-B and pEE12.

FIG. 45

Comparison of rat LO-CD2a with the humanized MEDI-507 antibody in aJurkat cell based binding assay.

FIG. 46

Comparison of the ability of rat LO-CD2a, humanized MEDI-507 antibody,and human IgG to inhibit a mixed lymphocyte reaction. The MLR responseof one responder/stimulator pair is shown as an example of relativeinhibition of the response by rat LO-CD2a or MEDI-507. Rat IgG2b orhuman IgG are the isotype controls.

FIG. 47

Comparison of rat LO-CD2a and the humanized MEDI-507 antibody in ahyporesponsiveness assay. Briefly, bulk 7 day MLR were performed in 6well plates in the presence of rat LO-CD2a, MEDI-507, or control rat orhuman IgG. The cultures were Ficolled to remove dead cells and rested inmedia for 3 to 4 days. The cells then were assessed for their ability toproliferate to a secondary challenge with the original allostimulator.The percent control of that proliferation relative to a rechallengedculture of a primary MLR which received no exogenous antibody is shown.This “media” treatment culture is taken as a 100% response.

FIG. 48

Comparison of the effect of rat LO-CD2a and humanized MEDI-507 antibodyon CD2 cell counts.

DETAILED DESCRIPTION

In accordance with an aspect of the present invention, there is provideda molecule (preferably a monoclonal antibody or fragment thereof) whichbinds to the same epitope (or a portion thereof) on human lymphocytes asthe monoclonal antibody produced by the cell line deposited as ATCCDeposit No. HB 11423. The antibody which is produced by the depositedcell line is hereinafter sometimes referred to as LO-CD2a. The term“molecule” or “antibody that binds to the same epitope as LO-CD2a”includes LO-CD2a. The term “LO-CD2a” includes the antibody produced bythe deposited cell line ATCC HB 11423 and those identical thereto whichmay be produced, for example, by recombinant technology.

The molecules or antibodies of the present invention inhibit humanT-cell activation and proliferation and Applicant has found that suchinhibition can be effected when adding the molecule or antibody eitherbefore or after an agent which stimulates T-cell activation.

The molecules or antibodies of the present invention have thecharacteristics of binding to an epitope of a CD2 antigen (CD2 positivehuman T-cells) but it is to be understood, however, that the ability ofsuch molecules or antibodies to inhibit T-cell activation orproliferation may or may not be effected through binding to CD2 positivecells, although Applicant presently believes that the mechanism ofaction involves binding of the molecule or antibody to CD2 positivecells.

In accordance with another aspect of the present invention there isprovided a method of preventing and/or inhibiting on-going immuneresponse in human patients through the administration to the patient ofan antibody, hereinafter referred to as LO-CD2a (or fragment orderivative thereof) or any molecule that mimics such antibody orderivative or fragment thereof.

A cell line which produces LO-CD2a, was deposited on Jul. 28, 1993, atthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md. 20852, and was given the ATCC accession number ATCC HB 11423. Suchantibody is a rat monoclonal antibody.

Although Applicants do not want to limit the invention to anytheoretical reasoning, it is believed that the mechanism which enablesthe monoclonal antibody of this invention to prevent or reduce theseverity of an immune response, and to inhibit the activation andproliferation of T-cells, is the fact that the LO-CD2a antibody eitherdecreases the density of CD2 expressed on T cell surfaces and thusdecreases the number of CD2⁺ T lymphocytes; and/or affects signaltransduction. It is believed that these mechanisms of action areresponsible for not only the prevention of immune response, but also thereduction in severity of on-going immune responses. In addition, theLO-CD2a antibody inhibits natural killer (NK) cell activity in vitro asexemplified herein. This is pertinent to the present invention since itis believed that a non-MHC restricted cytotoxic mechanism such as NKcell activity has been implicated in graft versus host disease.

In accordance with an aspect of the present invention there is provideda process for inhibiting initial or further activation and proliferationof T cells in a human patient by administering to the patient aneffective amount of a molecule (preferably an antibody) which binds tothe same epitope (or any part thereof) on human lymphocytes as theLO-CD2a antibody. The preferred molecule is LO-CD2a or a chimeric and/orhumanized form thereof. Such a molecule would, for example, contain thesame complementarity determining region (CDR) as the LO-CD2a antibody.

The term “inhibit” as used herein throughout this Applicant is intendedto mean prevention, or inhibition, or reduction in severity, orinduction of tolerance to, or reversal of graft rejection. The term“graft” as used herein for purposes of this application shall mean anyand all transplantation, including but not limited to, allograft andxenograft transplantation. Such transplantation may by way of exampleinclude, but not be limited to, transplantation of cells, bone marrow,tissue, solid-organ, bone, etc.

The term “immune response(s)” as used herein is intended to mean immuneresponses dependent upon T cell activation and proliferation whichincludes both cellular effects and T cell dependent antibodies which maybe elicited in response to, by way of example and not limitation: (i)grafts, (ii) graft versus host disease, and (iii) autoantigens resultingin autoimmune diseases, which by way of example include but are notlimited to rheumatoid arthritis, systemic lupus, multiple sclerosis,diabetes mellitus, etc.

The molecule employed in the present invention is one which binds to thesame epitope (or a part of that epitope) as the LO-CD2a monoclonalantibody. The term “binds to the same epitope as LO-CD2a monoclonalantibody” is intended to describe not only the LO-CD2a monoclonalantibody but also describes other antibodies, fragments or derivativesthereof or molecules which bind to the same such epitope as the LO-CD2amonoclonal antibody.

Such other antibodies include by way of example and not limitation rat,murine, porcine, bovine, human, chimeric, humanized antibodies, orfragments or derivatives thereof.

The term “derivative” as used herein means a chimeric or humanizedantibody, single chain antibody, bispecific antibody or other suchantibody which binds to the same epitope (or a portion thereof) asrecognized by the LO-CD2a monoclonal antibody.

The term “fragment” as used herein means a portion of an antibody, byway of example such portions of antibodies shall include but not belimited to CDR, Fab, or such other portions, which bind to the sameepitope or any portion thereof as recognized by LO-CD2a.

The term “antibody” as used herein includes polyclonal, monoclonalantibodies as well as antibody fragments, derivatives as well asantibodies prepared by recombinant techniques, such as chimeric orhumanized antibodies, single chain or bispecific antibodies which bindto the same epitope or a portion thereof as recognized by the monoclonalantibody LO-CD2a. The term “molecules” includes by way of example andnot limitation, peptides, oligonucleotides or other such compoundsderived from any source which mimic the antibody or binds to the sameepitope or a portion thereof as the antibody fragment or derivativethereof.

Another embodiment of the present invention provides for a method oftreating a patient who is to receive or has received a graft transplantwith an effective amount of at least one member selected from the groupconsisting of LO-CD2a antibody, or an antibody, or derivative orfragment thereof or molecules which bind to the same epitope (or aportion thereof) as the LO-CD2a antibody. The treatment is preferablyeffected with the whole or intact LO-CD2a antibody.

A monoclonal antibody of this invention as hereinabove described may beproduced by techniques known in the art such as described by Kohler andMilstein (Nature 256, Pg. 495-497, 1975) as well as the techniquesdisclosed herein. The preparation of a monoclonal LO-CD2a antibody isdescribed in more detail in Example 1 of this Application. Ashereinabove indicated LO-CD2a antibodies may also be produced byrecombinant techniques using procedures known in the art. Therecombinant antibody may also be in the form of a chimeric antibodywherein the variable regions of a LO-CD2a rat antibody are combined withthe constant region of an antibody of another species. Thus, forexample, the monoclonal antibody may be humanized by combining the CDRregions of a rat LO-CD2a monoclonal antibody with the V regionframeworks and constant regions of a human antibody to provide achimeric human-rat monoclonal antibody.

In one embodiment, the antibody is a humanized form of LO-CD2a antibodyconstructed from the constant regions of a human antibody, and theframework and CDR regions of the light and heavy chain variable regions,in which the framework regions of the light and heavy chain variableregions are derived from the framework regions of the light and heavychain variable region of a human antibody, and the CDR's are the ratLO-CD2a CDR's. In one embodiment, one or more amino acid residues of theframework regions of the light and heavy chain variable regions may beamino acid residues from the rat LO-CD2a framework regions. Suchresidues from the rat framework regions are retained in the humanizedantibody because such residues may maintain the binding specificity ofLO-CD2a. Thus, in producing a humanized antibody, in accordance with apreferred aspect of the invention, the CDR's of a human antibody arereplaced with the CDR's of LO-CD2a with the added factor that certainamino acids of the light chain variable portion of LO-CD2a in particularfrom FR1, FR2 and FR3 and certain amino acids of the heavy chainvariable portion of LO-CD2a in particular from FR-2 and FR-3 areretained in constructing the humanized antibody; i.e., the correspondingamino acids of the human framework are replaced with the noted aminoacids from the rat LO-CD2a framework. As noted with respect to FIG. 31amino acids 9, 12, 41, 42, 50, 51 and 82 in the framework of the lightchain variable region of rat LO-CD2a are retained and as noted in FIG.33, amino acids 47, 67, 70, 72, 76, 85 and 87 in the framework of theheavy chain variable region of rat LO-CD2a are retained in a humanizedantibody. A specific embodiment of the construction of such a humanizedantibody is given in Example 7 hereinbelow.

In another embodiment, in the humanized antibody, in the heavy chainvariable portion, certain amino acids from the FR1, FR2, and FR3 regionsare retained in constructing the humanized antibody. In particular, asnoted in FIG. 42, amino acids 47, 67, 70, 72, 76, 85, and 87, and one,two, three, or four of amino acids 12, 13, 28, and 48 in the frameworkof the heavy chain variable portion of rat LO-CD2a are retained in thehumanized antibody. In a preferred embodiment, amino acids 12, 13, 28,47, 48, 67, 70, 72, 76, 85, and 87 in the framework of the heavy chainvariable portion of rat LO-CD2a are retained in the humanized antibody.In another preferred embodiment, in addition to the retention of theabove mentioned amino acids in the framework of the heavy chain variableportion of rat LO-CD2a in the humanized antibody, the humanized antibodyfurther contains in the framework of the light chain variable region ofthe humanized antibody, one or more of amino acids 9, 12, 41, 42, 50,51, and 82 of the rat LO-CD2a light chain variable region as shown inFIG. 31. Preferably, each of amino acids 9, 12, 41, 42, 50, 51, and 82in the framework of the light chain variable portion of rat LO-CD2a areretained in the humanized antibody.

In a most preferred embodiment, the humanized antibody contains in theframework of the heavy chain variable region of the humanized antibody,amino acids 12, 13, 28, 47, 48, 67, 70, 72, 76, 85, and 87 of the ratLO-CD2a heavy chain variable region as shown in FIG. 42. The humanizedantibody also contains in the framework of the light chain variableregion of the humanized antibody, amino acids 9, 12, 41, 42, 50, 51, and82 of the rat LO-CD2a light chain variable region as shown in FIG. 31.Such antibody is sometimes hereinafter referred to as MEDI-507, theconstruction of which is detailed in Example 11 hereinbelow.

In another embodiment, the present invention is related to a chimericantibody comprised of a human constant region and the variable regionsfrom rat LO-CD2a and to the use thereof.

The antibody or molecule of this invention preferably: (i) binds to allT lymphocytes and also to null cells but not B lymphocytes as shown bytwo color staining of lymphocytes analyzed by flow cytometry (FIGS. 26and 27); (ii) binds to all T cells (as determined by staining with theanti-CD3 antibody Leu4), all CD4 and CD8 positive cells as defined byLeu3a and Leu2b antibodies respectively and some lymphocytes which areCD3 negative (null cells); (iv) binds to null cells as corroborated bythe staining of CD16 positive cells as detected with Leu11, a marker forNK cells. (FIG. 26); Staining of B cells, as defined by anti-CD19binding, was not seen with LO-CD2a. (FIG. 27). LO-CD2a antibody alsopreferably has the characteristic that the antibody binds to human nullcells, and by double staining has a higher intensity of staining tohuman cells that are both CD2+ and CD4+ than to human cells that areboth CD2+and CD16+, and has a higher intensity of staining of humancells that are both CD2+ and CD8+ than to human cells that are both CD2+and CD16+.

That Lo-CD2a binds to CD2 was confirmed by transiently expressing CD2 inCOS cells.

COS cells were transiently transfected with the πH3MCD2 plasmidcontaining the gene encoding for the entire CD2 molecule, as describedin Peterson A. and Seed B., Nature Volume 329, 10/29/87, pp 842-846.

Transfection was accomplished by the DEAE-dextran method. Cells wereharvested and stained with the anti-CD2 monoclonal antibody Leu5b(Becton-Dickinson) and LO-CD2a, with murine W632 an antibody to MHCclass I as a positive control for staining and with the correspondingisotype-matched controls. Specificity of the reactivity was confirmed byassessing binding of the same panel of monoclonal antibodies on COScells transfected with an irrelevant plasmid.

The staining pattern of these monoclonal antibodies on transientlyexpressed native CD2 (FIG. 28) indicates that transfection with CD2 ledto binding of both antibodies, supporting the ability of LO-CD2a to bindto CD2.

The preparation of LO-CD2a monoclonal antibody suitable for the purposesof the present invention should be apparent to those skilled in the artfrom the teachings herein.

An antibody or fragment or derivative thereof or molecule of the typehereinabove described may be administered in vivo in accordance with thepresent invention to inhibit the activation and proliferation ofT-cells, and decrease the density of CD2 expression on the cell surfaceand thereby reduce the number of CD2⁺ T lymphocytes.

Thus, for example, in an in vivo procedure, such LO-CD2a antibodies areadministered to prevent and/or inhibit immune response and therebyinhibit T cell activation and proliferation.

An antibody or fragment or derivative thereof or molecule of the typeherein above described may be administered ex vivo in accordance withthe present invention to decrease the density of CD2⁺ expression on thecell surface and thus reduce the number of CD2⁺ cells of the donorcells. By way of example and not limitation, in an ex vivo procedure,such antibodies or fragments or derivatives thereof or molecules wouldbe infused into donor bone marrow prior to transplantation to preventthe onset of graft versus host disease upon transplantation.

In such an in vivo or ex vivo technique, the antibody or fragment orderivative thereof or molecule will be administered in apharmaceutically acceptable carrier. As representative examples of suchcarriers, there may be mentioned normal saline solution, buffers, etc.Such pharmaceutical carriers are well known in the art and the selectionof a suitable carrier is deemed to be within the scope of those skilledin the art from the teachings contained herein.

The LO-CD2a antibody or other molecule of the present invention may beadministered in vivo intravenously or by intramuscular administration,etc.

As herein above indicated, LO-CD2a antibody or other molecule of thepresent invention is administered in vivo in an amount effective toinhibit graft rejection. The term “an effective amount” for purposes ofthis Application shall mean that amount of monoclonal antibody capableof producing the desired effect, i.e., the inhibition of graft rejectionor inhibition of the activation of T-cells. In general, such antibody isadministered in an amount of at least 1 mg. It is to be understood thatlower amounts could be used. In addition after the initial treatment,the herein above described amounts may be reduced for subsequenttreatments, if any. Thus the scope of the invention is not limited bysuch amounts.

In accordance-with the present embodiment, such antibodies areadministered in order to maintain the inhibition of T-cell activationand graft rejection. Thus, by way of example and not limitation, theantibody may be administered by intravenous infusion over a one to twohour period in amount of from about 1 mg/dose to about 50 mg/dose in aphysiologically acceptable carrier suspension once or twice a day for aperiod of from about eight days or more, as needed. Such treatment forgraft rejection is preferably started at, or immediately prior to, orshortly after transplantation or when graft rejection occurs. Thetreatment could be given once or twice a day for as little as one or twodays when started at the time of transplantation to induce a selectivehyporesponsive state to the transplant. Such treatment for autoimmunediseases with respect to the administration of the antibody or fragmentor derivative thereof or molecule in accordance with the presentinvention is begun when the attending physician has determined it isdesirable to inhibit a pathological immune response.

Thus, in accordance with an aspect of the present invention, byadministering an antibody in accordance with the invention at the timeof transplantation and in most cases for a short period thereafter therecan be induced a hyporesponsiveness to the transplanted tissue or organ,thereby to prevent or inhibit further episodes of rejection.

The techniques of the present invention for inhibiting the activation ofT-cells may be employed alone or in combination with other techniques,drugs or compounds for inhibiting the activation of T-cells orinhibiting graft rejection or graft versus host disease.

The invention will be further described with respect to the followingexamples, which are illustrative and which are not intended to limit thescope of the invention.

The cells, cultures, mAbs and mitogens used in the examples may beprepared and used by processes and procedures known and practiced in bythose of ordinary skill in the art. The following is an example of aprocess or procedure that may be used for the preparation and use of thecells, cultures, mAbs and mitogens used in the examples which follow.

Cells and Cultures

PBMC were obtained by Ficoll-Hypaque (Pharmacia, Sweden) sedimentationof heparinized blood obtained from the local Blood Donor Center.Isolated PBMC were resuspended in enriched medium: RPMI 1640 medium(Gibco, Belgium), supplemented with 100 U/ml penicillin, 100 μg/mlstreptomycin, 20 mM L-Glutamine, and 20% pooled human AB serum or 15%heat-inactivated fetal calf serum. PBMC were cultured at 1×10⁵cells/well in 96 U-well micro plates (Falcon) in a final volume of 200μl of culture medium/well. Bidirectional MLC were performed with 1×10⁵cells of each donor/well in the same volume of culture medium as notedabove. All cultures were made in triplicate. Eight hours before thetimes indicated in the results, cultures were pulse-labelled with 2.0μCi/well of 3_(H)-T (Amersham, Belgium; 247.9 GBq/mmol; 6.7 Ci/mmol) andthe radioisotope incorporated in cultures was quantified by liquidscintillation in a Betacounter (Beckman L5 6000 SE). The percentage ofinhibition was calculated as follows: % Inhibition=[1-(mean cpm oftested culture/mean cpm control culture)]×100. All results are expressedas the mean of three independent cultures. Standard deviation was alwaysless than 15% of the mean, except for those cases where these values areindicated on the graphics.

Cytofluorometric analyses were performed using a FACScan cytofluorograf(Becton Dickinson) with Hewlett-Packard hardware equipped with theConsort 30 program. Independent analysis of staining of lymphocytes andblast-cells was possible using differential gating as defined by sizeand granularity. 25,000 events were analyzed for each sample. In theseexperiments, LO-CD2a final concentration was 200 ng/ml, except whenindicated.

Mabs and Mitogen

LO-DRA and LO-Tact-1 (both FITC-labelled), are rat mAbs produced in ourlaboratory (op. cit. H. Bazin (Ed) 1990 p.287). LO-Tact-1 is directedagainst the p55 chain of the IL-2 receptor (op. cit. H. Bazin Immunol.1984 and Janszen, M., Buck, D. and Maino, V. C. in Leucocyte Typing IVWhite Cell Differentiation Antigens, W. Knapp (ED), Oxford UniversityPress, 1989, p.403). Mouse anti-human-CD2 and anti-CD3 mabs (Leu-5b andLeu-4a-FITC-labelled) were obtained from Becton Dickinson (Belgium).Mouse anti-human-CD4 or anti-human-CD8 mAbs (phcoerythrine-labelled),and mouse IgG FITC-or phcoerythrine-labelled (negative controls) wereobtained from Coulter. OKT3 (Ortho-Cilag, Belgium) was used at a finalconcentration of 100 ng/ml. Phytohemagglutinin A (PHA; Wellcome Labs,UK) and Concanavalin A (Con A; Calbiochem Co., USA) were used at a finalconcentration of 1 and 10 μg/ml, respectively.

Biotinylation of LO-CD2a. The concentration of purified LO-CD2a wasadjusted to 1 mg/ml in 0.1M sodium bicarbonate buffer, pH 8.4.NHS-biotin (Boehringer Mannheim 1008 960) was dissolved in DMSO at aconcentration of 1.5 mg/ml. For each MAB, 0.1 ml of NHS-biotin solutionwas added. The mixture was rotated for 2 hrs. at ambient temperature.The reaction was completed by adding 0.1 ml of 2M tris-HCL, pH 8.0, foreach ml of antibody (10 minutes at ambient temperature), followed by 1ml of 1% BSA in phosphate buffered saline (PBS) for each ml of antibody.To remove free biotin, the solution was dialyzed overnight at 4° C. in1000 volumes PBS. Both the biotinylation reaction and the conjugated mAbwere shielded from light by covering with aluminum foil.

Lysis of red blood cells (RBC). RBC were removed from whole blood bylysis with ammonium chloride. A 10×stock solution was prepared whichconsisted of 90g NH₄Cl, 10 g KHCO₃, 370 mg EDTA, and H₂O to a volume of100 mls. Forty mls of 1×ammonium chloride was added to each 10 mls ofblood and incubated for 10 min. at room temperature. The mixture wasthen centrifuged at 1200 rpm for 10 min and the pellet resuspended in 10ml PBS with 0.1% azide.

Staining of peripheral blood. Staining was carried out in round-bottom96 well cluster plates (Costar #3790) at 4° C. For single colorstaining, ten μl of mAb was appropriately diluted in PBS containing 0.2mg human immunoglobulin and added to each well. Red blood cell depletedblood was distributed into plates at a volume of 90 μl per well. Cellsand mAb were mixed by gentle tapping and incubated 30 min. Fifty μl ofcold PBS was added to each well and plates were centrifuged at 1900 rpmsfor 2 min. Supernatant was discarded by inversion and gentle flicking ofthe plate. Cells were dispersed by tapping the plate on the counter. Thewash procedure was repeated twice by adding 200 μl of cold PBS. Ten μlof a 1/20 dilution of goat F(ab′)₂ anti-rat 1g-FITC was added to thedispersed cells in each well and incubated for 30 min. in the dark.Cells were washed by the addition of 180 μl of cold PBS to each wellfollowed by centrifugation at 1900 rpms for 2 min. Supernatant wasdiscarded, cells dispersed, and 200 μl of cold 0.5% paraformaldehyde wasadded to each well. Cells were transferred to tubes (Falcon #2054) anddiluted to approximately 0.5 mls with 0.5% paraformaldehyde. Sampleswere evaluated on a Becton-Dickinson FACScan machine using LYSIS IIsoftware.

Dual color staining was carried out by a similar protocol. After cellswere incubated with the primary mAb and the FITC-conjugated anti-ratreagent, a 1/5 dilution of normal mouse serum was added to block anyremaining sites on the anti-rat reagent. Following a 15 min. incubation(no wash), 20 μl of a PE-labeled mAb specific for a known CD determinantwas added and incubated for 30 min. Cells were washed and fixed asdescribed for single staining.

EXAMPLE 1

LO-CD2a is a rat (IgG2b-Kappa) anti-CD2 monoclonal antibody produced andcharacterized in our laboratory as indicated elsewhere (See thefollowing references: Xia, H., Ravoet, A. M., Latinne, D., Ninanne, J.,De Bruyere, M., Sokal, G. and Bazin, H., in H. Bazin (Ed), RatHybridomas and Rat Monoclonal Antibodies, CRC Press, Inc., Boca Raton,Fla. 1990, p.309 and Ravoet, A. M. , Latinne, D., Seghers, J.,Manouvriez, P., Ninanne, J., DeBruyere, M., Bazin, H. and Sokal, G.- inH. Bazin (Ed) Rat Hybridomas and Rat Monoclonal Antibodies, CRC PressInc., Boca Raton, Fla., 1990, p. 287). LO-CD2a was purified from asciticfluid by immunoaffinity chromatography taking advantage of the allotypicdifference existing between the immunoglobulins of the rat receiving theproducing hybridoma and the mAb secreted by the latter (Bazin, H.,Cormont F. and DeClercq, L. J. Immunol. Method., 1984, 71:9). Itrecognizes the total population stained by the mouse mAb Leu-5b(FITC-labelled) FIG. 1 and roughly 90% of the population marked by themouse T11 (Rhodamine-labeled) mAb ((data not shown). The epitoperecognized by LO-CD2a on the CD2 molecule, is different from theepitopes recognized by the anti-CD2 mouse mAbs Leu-5b and T11 (FIG. 2).

EXAMPLE 2

LO-CD2a exhibits modulatory but not mitogenic effects on PBMC

In order to determine the effects of the rat mAb LO-CD2a on restinglymphocytes, PBMC were incubated in the presence of increasingconcentrations of this mAb. As can be seen in Appendix 1, PBMC incubatedfor 6 days in the presence of LO-CD2a show no significant variations inthe rate of ³H-T incorporation as compared with control cultures. Cellviability at the end of this period was variable but averaged around 80%as assessed by trypan blue exclusion. When resting PBMC were incubatedin the presence of LO-CD2a, there was no significant variation in thephenotypic expression of several membrane markers, as assessed by flowcytometry. Cellular markers of resting mature T-cells (such as CD3, CD4and CD8) show the same pattern of variation during 6 days of culture inthe presence or in the absence of LO-CD2a, and activation molecules suchas CD25 (IL-2R/p55) are not expressed in these experimental conditionsor are not modified by LO-CD2a as is the case of DR antigenicdeterminants. (FIG. 3)

When PBMC were incubated for 6 days in the presence of LO-CD2a, asignificant decrease was observed in the percentage of Leu-5b+gatedlymphocytes. (FIG. 4) The percentage of CD4-and CD8-lymphocytes is notaffected during a 6-day period of cultures by the presence of LO-CD2a,indicating that the observed decrease of CD2-bearing lymphocytes cannotbe attributed to an elimination of these cells but rather to adisappearance of the CD2 molecule or to a conformational change in thisglycoprotein produced by the binding of LO-CD2a.

In order to verify if the observed decrease in Leu-5b-lymphocytes wasdue to a conformational change of CD2 or to a disappearance(internalization or release) of this molecule after the binding ofLO-CD2a, PBMC were cultured in the presence of 500 ng/ml of LO-CD2a andanalyzed through 6 days in flow cytometry using Leu-5b (FITC-labelled),T11-RD1 (Rhodamine-labelled) and MARK-3 (FITC-labelled). As shown inFIG. 5 a, Leu-5b or T11 mabs are not able to bind to PBMC after 2 to 4days of culture in the presence of LO-CD2a. Under these conditions, themouse anti-rat kappa chain mAb MARK-3 labelled 50% of cells at day 6 ofculture indicating that only 35% of the original CD2-bearing cells showno LO-CD2a on their surfaces, yet Leu-5b-FITC and T11-RD1 staining havedecreased markedly at day 2. This suggests that a conformationalalteration of CD2 rendering the epitope of Leu5b and T11 unavailable forbinding occurs in response to LO-CD2a.

The analysis of the mean fluorescence of CD2+ cells indicated that thedensity of expression of this marker decreased with time in the presenceof LO-CD2a. The same phenomenon was observed whether Leu-5bFITC-labelled or LO-CD2a (revealed by MARK-1 FITC-labelled) were used todetect the CD2+ lymphocytes. Aliquots of the same PBMC were cultured inparallel in the presence of Leu-5b (commercially available mAb, dialyzedagainst PBS, 1:2 final dilution in culture medium). As shown in FIG. 5b, in those experimental conditions all the CD2-bearing cells are coatedby the Leu-5b mAb (as revealed by goat anti-mouse-FITC). Staining byT11-RD1 was markedly reduced, whereas a smaller, slower decrease wasobserved in the percentage of cells presenting the epitope recognized bythe LO-CD2a-FITC mAb. Taken together these results indicate that CD2molecules have partially changed their conformation in response toLO-CD2a, and that a slow modulation of CD2/LO-CD2a occurs.

LO-CD2a Inhibits MLR

When MLC were performed (over a period of 6 days) in the presence ofincreasing concentrations of rat mAb, a significant inhibition of theMLR (as measured by ³H-Thymidine (³H-T)-incorporation), was observed atconcentrations of mAb as low as 125 ng/ml. In FIG. 6 a, we show atypical example of a dose-response curve of MLR inhibition by LO-CD2a.As can be seen in this FIG. 6 a, LO-CD2a induces 80% inhibition of MLR(6 days of culture) at 250 ng/ml and this percentage of inhibitionremains almost constant or higher than 80% over a wide range ofconcentrations (0.25 to 5.0 μg/ml of mAb). FIG. 6 b shows a time-courseof the inhibitory effects of different concentrations of LO-CD2a on MLRfrom day 0 to day 6 of culture. A typical example of ³H-T-incorporationon MLC (in the presence or in the absence of LO-CD2a) is shown in FIG. 6c, where LO-CD2a was added at a final concentration of 200 ng/ml.

In FIG. 6 d we show the effects of LO-CD2a on MLR, when this mAb (at 200ng/ml) is added at varying times after initiation of MLC. More than 90%inhibition of MLR (as measured by ³H-T incorporation) is obtained whenthis mAb is added at day 0, and this inhibitory effect is still present(45% inhibition in this example) when LO-CD2a is added 4 days after thebeginning of MLC. Similar results (not shown) were obtained with higherconcentrations (from 0.20 to 5.0 μg/ml) of LO-CD2a.

LO-CD2a Blocks the Pathway of IL-2R Expression

When cytofluorograph analyses were performed on the lymphoblast subsetof a MLC (FIG. 7 a and b), the following observations were made: a) thenumber of blast cells (around 300-500 blast cells of 25,000 eventsanalyzed) already present at initiation of MLC rose sharply from day 4to day 6 in control cultures (more than 1200 blast cells from 25,000events analyzed); b) in MLC performed in the presence of LO-CD2a, therewas no significant variation in the number of blast cells during thewhole period of culture and at day 6 the number of blast cells is alwayslower or nearly the same as the initial number of blasts at day 0 (FIG.7 a); c) the percentage of CD25 blasts rose sharply among cellsincubated without LO-CD2a (FIG. 7 b); d) this percentage remains below20% in the small number of blasts from the MLC incubated in the presenceof mAb (FIG. 7 b), and the mean fluorescence (as a measure of CD25expression) decreased by 75% as compared with blasts present in controlcultures (results not shown); e) in the absence of mAb the percentage ofCD3-blasts remains constant during the first 4 days of culture (FIG. 7b) and on day 6 the percentage of CD3-cells increased to 90%, while inthe presence of LO-CD2a the percentage of CD3-rises slowly to reach onlyabout 45% at day 6. These results indicate that the presence of LO-CD2ainhibits the entrance of these cells in the pathway of activationcharacterized by the expression of IL-2 receptor (CD25). The number ofCD2+ blasts remains constant or decreases in the presence of LO-CD2a,and the density of expression of this membrane marker is stronglydiminished under these conditions (data not shown).

When phenotypic analyses were performed through 6 days on the resting(non blast) lymphocyte subset of MLC, results similar to those describedin FIG. 3 were obtained: in the presence of LO-CD2a, no significantvariation could be detected in the percentage of CD3+, CD4+ or CD8+lymphocytes, as compared with control cultures; no CD25 expression(activation marker) could be detected whether in the presence or in theabsence of LO-CD2a during 6 days of culture (FIG. 8 a). These resultssuggest that LO-CD2a has a very weak, if any, effect on the restingsubset of T-lymphocytes in MLC; that is to say, in T-cells not committedin the process of activation. At the same time, as shown in FIG. 8 b,LO-CD2a induces a significant decrease in the percentage of CD2+lymphocytes during MLC. These results suggest that in both theunstimulated cultures (FIG. 5) and in the MLC, the effect of LO-CD2a isto reduce the expression of CD2 and/or to induce a conformational changein its structure.

LO-CD2a can block the pathways of T-cell activation dependent on theTcR/CD3 complex or on mitogen receptors.

When LO-CD2a was added to mitogen-activated PBMC, a significantinhibition of ³H-T incorporation was observed. In one of threeexperiments of PBMC incubated with mitogens (OKT3, ConA and PHA) in thepresence or in the absence of LO-CD2a added either at time 0 or 1 hourafter the start of cultures. In the first case, mitogens were added 1hour later. When LO-CD2a was added 1 hour after the initiation ofculture, mitogens were added at time 0. This was done in order to knowwhether, preincubation of PBMC with mitogens or LO-CD2a could triggerevents that could be affected by the addition of the second reagent.Cultures were harvested at 96 h, after a pulse-labelling (6 h) with³H-T. More than 50% inhibition of ³H-T. incorporation was observed inthe presence of LO-CD2a, whether it is added first or after mitogens.(FIG. 9) The same effect was observed when cells were harvested 4 daysafter the onset of MLC and exposed to mitogens (results not shown). Adrastic decrease in ³H-T incorporation was observed two days after theonset of MLC, in those cultures receiving both the mitogen and LO-CD2a,as compared with the same cultures receiving only mitogen (results notshown). Preincubation of MLC with LO-CD2a before addition of mitogen,lowered the ³H-T-uptake to values comparable with MLC without OKT3.

LO-CD2a was also able to inhibit mitogen induced proliferation if addedone day after the initiation of mitogen induced proliferation. Theresults of experiments performed with two donors are shown in FIG. 10.PMBC were incubated along with mitogens (OKT3, ConA and PHA). In theseexperiments, LO-CD2a was added either at time 0 (Day 0), 24 h (Day 1) or48 h (Day 2) after the start of the cultures. The inhibition ofproliferation in response to OKT3 and ConA by LO-CD2a was significant ifadded 24 hours after the addition of mitogen at time 0.

EXAMPLE 3

Inhibition of Natural Killer Cell (NK) Activity.

PBMC were isolated from heparinized blood by Ficoll HypaqueSedimentation. After washing, the effector cells, suspended in enrichedmedium, were incubated overnight at a concentration of 1×10⁶/ml in aFalcon plate to eliminate the monocytes (by adherence).

The target cells (K562 cell line) were labeled by overnight incubationwith ⁵¹chromium ⁵¹Cr (0.9 ml of a cell suspension at 3×10⁶/ml+0.02 mlfrom a solution of 5 mCi/ml⁵¹Cr, Amersham).

After a 16-hour incubation, effector and target cells were washed fourtimes, counted and incubated in a 96 V bottom microplate at differentE/T ratios: 200/1 (100 ul of a suspension of 4×10⁶/ml effector cellswith 100 ul of 2×10⁴/ml target cells) 100/1, 50/1 and 25/1.

After a four-hour incubation, the ⁵¹Cr release was measured by counting100 μl supernatant from each well in a gamma counter.

Maximum (target cells+HCLIN) and spontaneous release (targetcells+enriched medium) were used to calculate the specific lysis:${\%\quad{Specific}\quad{lysis}} = {\frac{\text{test-spontaneous~~release}}{\text{maximum-spontaneous~~release}} \times 100\%}$

Inclusion of LO-CD2a at 5, 1 and 0.5 ug/ml in the NK assay with twonormal donors (FIGS. 10 a and 10 b) led to an inhibition of cytotoxicityof approximately 50% with all tested concentrations of antibody and overall tested E/T ratios. This is in comparison with essentially completeinhibition of proliferation in the MLR at doses at or above 0.25 ug/ml.

EXAMPLE 4 IN VIVO STUDIES IN NON-HUMAN PRIMATES Material and Methods

Monoclonal Antibodies

MARK3-FITC is a mouse mAb directed against the rat Ig kappa 1b allotypeconjugated with FITC. MARG2b-biotin is a mouse anti-rat IgG2bimmunoglobulin mAb conjugated with biotin. These two mAbs were producedand labeled in our laboratory. For immunofluorescent tests they wereused at a final concentration of 2.5 μg/ml. Leu-5b-FITC(Becton-Dickinson) and T11 Rhodamine (COULTER) are two mouse anti-humanCD2 mAbs. T4- and T8-Rhodamine-labeled (COULTER) are mouse anti-humanCD4 and CD8 mAbs, respectively.

Phenotype Analysis

Anti-human T-cell mAbs (anti-CD2, -CD4, -CD8, see above) were added to100 μl samples of whole blood and incubated at 4° C. for 45 min. Redblood cells were lysed with a Tris-buffered ammonium chloride-lysingbuffer (144 mM NH₄CL₁/17 mM Tris, pH 7.2) and lymphocytes were washedwith PBS/2% FCS/0.2% NaN3. For detection of non-labeled mAbs, a secondmAb (FITC- or biotin-conjugate) was added to a final concentration of2.5 μg/ml. After 45 min. incubation at 4° C., cells were washed withPBS/FCS/NaN3. For biotinylated mAbs, a further incubation (15 min) withStreptavidin-Phycoerythrin conjugate was done. Labeled human or monkeylymphocytes were resuspended in a 2% formalin solution and analyzed in aFACSan cytofluorometer (Becton-Dickinson) equipped with the lysis IIprogram for gating lymphocytes as a function of size-vs-granularity. Asa control for nonspecific staining, aliquots of cells were incubatedwith FITC-or Phycoerythrin-conjugated mouse Igs (Coulter).

Level of Circulating Abs

LO-CD2a in serum was quantified by ELISA using a mouse anti-rat lgG2bmAb (MARG2b-8, produced in our laboratory ) as first layer (coating) anda mouse anti-rat kappa chain (MARK-3) mAb coupled to horseradishperoxidase for detection. Briefly, microtiter plates (Falcon) wereincubated overnight with 100 μl/well of MARG2b-8 (5μg/ml) and unoccupiedsites on plastic were saturated with PBS containing 5% powdered milk(bovine). After 1 h incubation at room temperature, plates were washedwith PBS with 0.1% Tween-20, and incubated 1 h with 100 μl/well ofdiluted monkey or human serum. After washing out unbound material,plates were incubated 1 h with 100 μl/well MARK3-peroxidase (2 μg/ml inPBS). After washing again, plates were incubated with OPD(o-phenylenediamine dihydrochloride, 0.4 mg/ml, Sigma Chemicals), incitrate-phosphate buffer containing 0.03% H₂O₂. The colored reactionproduct was detected at 492 nm. A standard curve was made in parallelwith a known concentration of purified LO-CD2a serially diluted in apool of control monkey or human serum.

The detection of monkey or human anti LO-CD2a antibodies was performedby ELISA using 96 well microtiter plates coated with LO-CD2a (5 μg/ml).Anti-LO-CD2a human or monkey antibodies bound on the plates, wererevealed by horse-radish peroxidase labeled rat anti-human IgM (LO-HM-7)or IgG (LO-HG-22) mAbs.

A. Cynomolgus Monkeys

One Cynomolgus monkey received 10 mg/day of LO-CD2a for threeconsecutive days. The monoclonal antibody was well tolerated.

Lymphocyte depletion was observed after the first injection but a verylittle additional depletion was obtained after the 2d and 3d injections.

The second monkey received 20 mg/d for 10 days. The mAb was also welltolerated. No side effects were observed after dosing in that theanimals were active, alert, eating well with no evidence of nausea orgastrointestinal disturbance.

The lymphocyte counts and cell populations in the second monkey aresummarized in FIGS. 12 and 13. The NK activity was slightly reducedafter the 10 injections (FIG. 14). The circulating levels of MAb werevery high (FIG. 15) and immunization occurred at the end of thetreatment (FIG. 16).

B. Baboon

The experiment described here was undertaken to determine the toleranceof a baboon to LO-CD2a, to analyze the effects of this mAb on some ofthe membrane markers of baboon lymphocytes and to determine thehalf-life of LO-CD2a in serum.

Staining of baboon cells with LO-CD2a results in <20% positive cells ata mean fluorescence intensity significantly lower than that of stainedhuman cells. This staining pattern may reflect weak cross-reactivity orbinding via Fc interaction with baboon cells.

The study was done on a male baboon (papio mormon) weighing 8.8 Kg.Before each injection of LO-CD2a the monkey was anesthetized; the firsttime with Ketaler (2 ml) and Prazine (0.5 ml), the second time withKetaler only and the subsequent times with ketaler and Prazine (0.3 ml).LO-CD2a was injected intravenously (i.v. in 10 min.), diluted in 100 mlof physiological saline. For phenotypic analysis of lymphocytes andmeasurement of circulating antibodies (injected LO-CD2a, newly formedanti-LO-CD2a antibodies, and preexisting cross-reacting baboonanti-LO-CD2a antibodies), blood samples (10 ml) were taken in two tubes.The tube for lymphocyte typing contained EDTA. Samples were taken priorto the first treatment to determine baseline levels.

The first dose (10 mg) of LO-CD2a was administered on day 0 of thestudy; the four following doses (10 mg/dose) were administered on days7, 8, 9, and 10. Blood samples were taken a few minutes after eachLO-CD2a dose. On days 7 and 9 a supplementary blood sample (in anEDTA-containing tube) was also taken before the LO-CD2a injections.Blood samples were taken on days 1, 2, 11, 12, 13, 16 and 24.

No abnormal reactions in activity or feeding habits were observed duringLO-CD2a injections or throughout the period of study. The weight of theanimal remained around the 8.8 Kg measured at day 0 (see table below).

Day 0 7 8 9 10 12 13 16 24 Weight 8.8 8.9 9.1 9.1 8.8 9.0 9.1 8.9 8.9

ANALYSIS OF PHENOTYPE AND CIRCULATING mAb

The fluorescent staining of this baboon's peripheral blood lymphocytesrevealed some interesting features: a) Under the effect of LO-CD2a theCD2-positive subset of lymphocytes decreased significantly (as revealedby two different anti-CD2 mAbs) at the end of the 5th dose of LO-CD2a,that is at the time of a maximal accumulation of mAb in blood (see FIGS.17 a and 17 b). Given that the CD4+ and the CD8⁺ subsets of lymphocytesdo not decrease during this period (FIG. 19), and because the CD4+ andCD8+ cells comprise most of CD2 bearing lymphocytes, the decrease inCD2⁺ cells indicates that it is the membrane marker expression that isdecreasing or altered in conformation, rather than the lymphocytes.

As can be seen in (FIG. 17 a), a slight decrease of CD2+ (Leu-5b⁺ orT11⁺) positive lymphocytes is observed after the first dose of LO-CD2a.Two days after this first dose, the level of CD2 positive cells (Leu 5b⁺of T11⁺) rose to the starting values.

At the end of the four 24-hour spaced doses of LO-CD2a (days 7-10) thepercentage of CD2 positive cells (Leu 5b⁺ or T11⁺) decreased sharply andbegan to rise slowly 3 days after the end of the LO-CD2a administration.b) At the same time, the percentage of LO-CD2a positive cells, that is,the percentage of cells bound by the circulating mAb rose to 22% afterthe 2nd dose of LO-CD2a (day 7) and then decreased as did the CD2⁺ cellsrevealed by the anti-CD2 mAbs Leu-5b and T11 (FIG. 17). The decrease ofLO-CD2a+ cells was revealed by the MARK-3FITC mAb (FIG. 11).

The decrease of LO-CD2a+ cells was determined by detection of theLO-CD2a present on cells as detected by MARK3-FITC or by theMARG2b-8-biotin conjugated mAbs (FIG. 18 a). The same phenomenon wasobserved if cells were first incubated with LO-CD2a at 2.5 μg/ml tosaturate all the sites unoccupied by circulating mAb. LO-CD2a wasdetected by MARK-3-FITC or MARG2b-8-biotin (FIG. 18). c) As can be seenin (FIG. 19), the T4 positive subset of baboon lymphocytes showed amoderate rise during days 9 to 12 after which the percentage of T4⁺lymphocytes returned to its initial value. Concomitantly with the risein T4⁺ cells, the percentage of T8 positive lymphocytes rose from day 9to day 11. After that day this percentage returned to initial values. d)The levels of circulating mAb LO-CD2a decreased to background values 3days after the first injection (see FIG. 17 b). When LO-CD2a is appliedin four short-time spaced doses (days 7 to 10), the levels of serumLO-CD2a (around 3.7 mg/ml, maximal value in this period) decreasedslowly after the last dose (days 10 to 16), indicating a relatively longhalf-life of the Ab in this animal model. No baboon anti-LO-CD2aantibodies were detected in the blood samples collected on days 11, 12,13, 16 and 24.

CONCLUSION

LO-CD2a seems to be well tolerated by,non-human primates, asdemonstrated by the absence of apparent reactions in cynomolgus monkeybaboon. LO-CD2a seems to have a relatively long half-life in the baboon.Twenty-four hours after the first dose of LO-CD2a (day 1 in FIG. 24 b),50% of the maximal detectable level of MAb was still present in serum.Three days after the last dose of LO-CD2a (day 13 in FIG. 24 b), 50% ofthe maximal detectable level of mAb was still present in serum.

Although the staining pattern in non-human primates is not consistentwith that observed for CD2 on human cells, the decrease in thepercentage of CD2 positive lymphocytes followed by a slow rise of thispercentage of cells is similar to that observed in human PBMCmononuclear cells cultured in the presence of LO-CD2a.

EXAMPLE 5 Patients Treated with LO-CD2a Patients Treated with LO-CD2a ona Compassionate Basis PATIENT #1 (Mb.E.)

This was a female patient with chronic pyelonephritis, who was treatedwith a renal allograft for end-stage renal failure. A rejection crisisoccurred and was treated with 10 days of OKT3. The creatinine leveldropped from 2 to 1.4 mg/dl. Approximately four (4) months later arejection crisis was diagnosed by a creatinine level of 2 mg/dl and abiopsy indicating moderate rejection. The patient was treated with 1.5 gSolumedrol and a course of ATG for the following eight (8) days at whichtime the creatinine level was 1.65 mg/dl. Seven days after treatment abiopsy was performed and indicated cellular rejection and moderatevascular rejection. Two days after the biopsy (day 0) the patient wasanuric with a creatinine level of 2.4 mg/dl. That same day the patientreceived 10 mg of LO-CD2a, 1.5 g of the corticosteroid Solumedrol, plus1 g Polarimin (an anti-histamine) and 1 g Dafalgan (acetaminophen). Thetreatment with the corticosteroid, dexchlorpheniramine and acetaminophenis referred to by the transplant community as “coverage”. No sideeffects were noted. By the end of 23 hours, the patient produced 700 mlof urine and the creatine was 2.72 mg/dl. For the next 9 days shereceived 10 mg/day of LO-CD2a. The patient left the hospital without afollow-up biopsy at that time, Day 11.

Measurement of serum creatinine level during ATG treatment and duringthe following LO-CD2a treatment indicated the creatinine level rosedespite ATG treatment and fell and stabilized with LO-CD2a (FIG. 27).

The leukocyte count fell from a high of 10,000 to 2,000 during thetreatment with LO-CD2a and continued to fall until the last measurementon day 21 (FIG. 20). The lymphocyte count was low and variable duringthe period of observation.

The serum levels of LO-CD2a rose to peaks of 2.0-3.0 μg/ml immediatelyfollowing each treatment and fell to lows of approximately 1.0 μg/mlbetween each treatment (FIG. 21). With the last treatment on day 9, thelevel fell by 50% in 24 hours and to 0 by day 14. This patient returnedto the clinic on day 40.

The patient's creatinine level was 2.27 on day 40, 2.48 on day 50 androse to 3.11 by day 66 at which point a biopsy was obtained, with theinitial report consistent with severe cellular rejection andinterstitial hemorrhage (see below). The patient was treated with 150Rof irradiation to the kidney, 3×125 mg Solumedrol, while continuing onmaintenance therapy of cyclosporin plus 12.5 mg/day of steroids. Thecreatinine level continued to rise during the subsequent period. On day70 the creatinine level was 3.3; day 80, 5.63; day 84, 8.35. By day 86the creatinine level was 10.8 and a transplant nephrectomy was performedon day 88. This patient's compliance with maintenance immunosuppressionduring the period between her discharge on day 10 and her biopsy on day66 is in question and the loss of the kidney despite the evidentlysuccessful rescue must factor in the uncertain compliance.

(ii) PATIENT #2

The patient was a 38 year old male who was Hepatitis C⁺. He had receiveda renal allograft for the treatment of end stage renal failure due tochronic interstitial nephropathy. One year and three months later, heunderwent a transplant nephrectomy due to acute cellular and vascularrejection resistant to a course of OKT3.

One year and ten months from the transplant nephrectomy, he received asecond renal allograft. Three days later his creatinine level was 1.4mg/dl. Three days later he received 500mg Solumedrol; the patient'screatinine later that day was 1.8 mg/dl. On the following day hereceived 500 mg of Solumedrol; creatinine was 3.25 mg/dl. The followingday he received 500 mg Solumedrol; his creatinine was 2.95 mg/dl. Threedays later his creatine level was 2.3 mg/dl and he underwent a biopsywhich demonstrated 3 plus cellular rejection. Three days later hereceived 10 mg LO-CD2a, plus 200 mg Solumedrol. Polaramine and Dalfagan.Side effects observed were limited to sleepiness; no hyperthermia orhypertension were noted. For the next 9 days he received dailytreatments of 10 mg of LO-CD2a. The day following the end of suchtreatments a biopsy showed no signs of rejection.

The patient tolerated the course of LO-CD2a well with no evidence ofclinical side effects, including no fever or hypertension with any dose.Routine hematological and clinical chemistry laboratory tests (includingLFTS) obtained during the course of treatment demonstrated noalterations attributable to the administration of the antibody, exceptfor a decrease in the lymphocyte count from 290/cubic mm to a low of100/cubic mm and the reduction in creatinine level associated withresolution of the rejection crisis (from 2.7 mg/dl at the initiation oftreatment with LO-CD2a to 1.10 at the end of the course).

FIG. 22 shows the serum creatinine level of this patient, as fallingfrom 2.5 to approximately 1.0 on the days following treatment withLO-CD2a. The patient was lymphopenic prior to and during treatment andthe leukocyte count showed no dramatic alteration with treatment (FIG.23). In this patient the serum levels of LO-CD2a did not rise above 2.0μg/ml after each treatment and fell to lows of 1.0 to less than 0.25μg/ml (FIG. 24). Eight months after the first treatment with LO-CD2a thepatient was doing well with normal renal function and no evidence ofrecurrent rejection.

Patient 2—Biopsy #1—Diagnosis: Indeterminate.

The biopsy contained about 20 glomeruli which are unremarkable. Therewas a sparse mononuclear infiltrate with a minor degree of interstitialedema. Only minor degrees of tubular invasion were found and no vascularlesions. These findings are insufficient for the diagnosis of acutecellular rejection. There were rare mononuclear cells in small arteries,which were suspicious, but did not meet the criteria for the diagnosisof rejection.

Patient 2—Biopsy #2 Approximately 2 weeks after the first biopsy—Nodiagnostic abnormality recognized.

The biopsy looked similar to the previous biopsy and contained about 10glomeruli. The infiltrate was very sparse and no vascular lesions wereidentified.

(iii) PATIENT #3

The patient was a 19 year old with von Willebrand disease who received arenal allograft for the treatment of end stage renal failure due tochronic pyelonephritis. The transplant was removed on 17 days later dueto acute vascular rejection with secondary hypertension after failure ofa 6 day course of OKT3.

Five and one-half months later he received a second renal allograft. Tendays later his creatinine level was 6 mg/dl. The next day the creatininelevel was 7 mg/dl and a biopsy indicated 3 plus cellular rejection andvascular rejection (proliferative endarteritis without necrosis orthrombosis). That same day he received 10 mg of LO-CD2a, 40 mgSolumedrol and Polaramine and Dafalgan. No side effects were observed.For the next 9 days he received daily treatments of 10 mg of LO-CD2a,with no other drugs and no side effects. Two days after completion ofthe treatment, his creatinine level was 1.75 mg/dl and a biopsyindicated no sign of acute rejection, with interstitial necrosis and onefocal spot of chronic rejection.

No clinical side effects (alteration in BP or temperature) wereobserved. Routine hematological and clinical chemistry laboratory tests(including LFTs) showed no changes attributable to administration of theantibody except the decrease in creatinine level associated with theresolution of the rejection crisis (from 7.10 mg/dl on the initiation oftreatment with LO-CD2a to 1.75 mg/dl at the end of the 10 day course).The lymphocyte count was 340/cubic mm prior to treatment and fell to alow of 220/cubic mm during treatment, rose to 690/cubic mm 9 days aftercessation of treatment with the LO-CD2a and had risen to 1000/cubic mm23 days after the end of treatment.

The leukocyte count in this patient was not significantly altered bytreatment (FIG. 25). The serum creatinine level fell dramatically withtreatment (FIG. 25). Seven months after the first treatment with LO-CD2athe patient was doing well with normal renal function and no evidence ofrecurrent rejection.

Patient 3—Biopsy #1—Diagnosis: Severe cellular rejection affecting smallarteries and to a lesser degree the interstitium and glomeruli.

An arcuate sized artery showed a marked mononuclear infiltration of theintima with disruption of the elastica. There was sparse infiltrate inthe interstitium, which occasionally invaded tubules. The interstitiumshowed diffuse, mild interstitial edema. There were about 7 glomerulipresent. These show hypercellularity with mononuclear cells andendothelial swelling. Overall, this pattern was diagnostic of severe,acute cellular rejection.

Patient 3—Biopsy #2 Approximately 2 weeks after the firstbiopsy—Diagnosis: Consistent with treated rejection.

The biopsy showed a few small arteries, which show intimal fibrosissometimes with a mucoid material but a very minimal cellular infiltrate.The interstitium showed a fine diffuse fibrosis and a minimalmononuclear infiltrate. Tubules were locally atrophic but otherwiseunremarkable. There was no evidence of active cellular rejection.

No subsequent rejection episodes have been reported during 28 months offollow-up of the second and third patients who remained compliant withtheir immunosuppressive therapy.

(iv) PATIENT #4

The patient who was suffering from severe graft versus host disease(severe skin, gut, renal and CNS toxicity resistant to high doseprednisone) after an allogeneic bone marrow transplant received 12 daysof LO-CD2a at 10 mg/day. His symptoms improved; renal function returnedto normal, diarrhea ceased, skin improved and confusion resolved. Fourdays after the antibody was stopped the symptoms recurred and thepatient died despite the initiation of a second course of antibody.

LO-CD2a thus could be used to reverse ongoing immune responses toforeign tissues (allogeneic and xenogeneic, since it inhibits the xenoMLR as well as the allo MLR). The antibody would be given by i.v.infusion once or twice a day for 10 days to 14 days. It may also be usedprophylactically to prevent activation of T cells as part of theinduction protocol immediately following organ transplantation.

In a second experiment, a Phase I safety, pharmacokinetic anddose-finding clinical trial was initiated in renal allograft recipientsundergoing an initial biopsy proven acute rejection episode. Theantibody preparation used in this trial is LO-CD2a, produced in cellculture.

Compassionate use of the antibody showed no side effects and suggestedefficacy in reversing acute graft rejection at a dose of 10 mg/day for10 days. Preclinical studies with chimpanzees suggested that similar invivo effects were observed with doses equivalent to human doses rangingfrom 0.1-100 mg/day with no obvious adverse effects. Therefore anobjective of this Phase I trial was to investigate decreasing doselevels of LO-CD2a starting at 10 mg/day for ten days (with an optionalextension for an additional five days) in order to obtain indications ofthe minimal effective dose. Because no side effects had been observedwith steroid ‘coverage’ in the compassionate use patients describedabove, it was decided to administer the antibody with no steroidcoverage and only minimal pretreatment with analgesics andantihistamine. This was done to characterize predictably the sideeffects induced by LO-CD2a.

Eleven patients have been enrolled under this protocol to date. Fourpatients were treated with 10 mg/day, five patients were treated at 5mg/day and two patients were treated with 2.5 mg/day.

Among the eleven patients enrolled, nine patients experienced reversalor partial reversal of acute rejection confirmed by biopsy: all fourpatients treated with LO-CD2a at 10 mg/day, three of the patents treatedwith 5 mg/day, and both patients treated with 2.5 mg/day. One of thepatients treated with 5 mg/day (Patient 8, see below) withdrew from thestudy voluntarily after the first dose and a second patient treated with5 mg (Patient 9) showed a poor response.

Table 1 summarizes results obtained with renal rejection patientstreated with LO-CD2a under this protocol.

TABLE 1 Dose Number of Rejection Recurrent Patient (mg/day) rejectionsReversed? Rejection? #1 10 1st Yes No (13 mo.) #2 10 1st YesYes_(-non compliant) (13 mo.) #3 10 1st Yes Yes (1 mo.) #4 10 1st Yes No#5 5 1st Yes No (10 mo.) #6 5 1st Yes No (10 mo.) #7 5 1st Yes No (9mo.) #8 5 1st N/A¹ N/A #9 5 1st No N/A #10 2.5 1st Yes Yes (5 mo.) #112.5 1st Yes No (4 mo.) ¹withdrew at patient's request

Adverse events observed during treatment with LO-CD2a without steroidcoverage included nausea, vomiting, fever, chills, and hypertension,possibly as a result of a transient release of cytokines. The majority(greater than 70%) of these events were observed during administrationof the first dose and were of limited extent and duration. For example,no fevers greater than 40° C. were observed and most of the eventsresolved within hours of onset. No hypotension or severe diarrhea wasobserved. There was no clear relationship between the intensity andincidence of these events and antibody dose. Despite the obviousdiscomfort of these symptoms, no events required emergent resuscitativemeasures.

In a third experiment, ten patients with acute renal allograft rejectionwere treated on a compassionate basis without steroid coverage withLO-CD2a as follows:

Two were treated with 10 mg/day, four were treated with 5 mg/day, andfour were treated with 2.5 mg/day. All compassionate use patientstreated with 10 mg/day and 5 mg/day, and all but one at treated with 2.5mg/day showed evidence of complete or partial resolution of rejection bybiopsy and other clinical signs. The adverse event profile with thesecompassionate use patients resembled that seen hereinabove withpredominantly first-dose symptoms of limited duration and extent.

In a fourth experiment, six patients with steroid resistant graft versushost disease, and one liver transplant recipient have received LO-CD2aon a compassionate use basis. No adverse events were reported for thesepatients. Brief narratives for these patients follow.

All six GvHD patients were treated by 10 consecutive daily doses of 10mg of LO-CD2a, administered intravenously over one hour. Concomitantly,cyclosporin and steroids were continued. All but one patient showed animprovement over their GvHD symptoms. The resolution of the GvHDsymptoms began on day 3-6 after starting mAb therapy. A progression ofsigns of hepatic GvHD under mAb therapy has been observed in twopatients. Relapse of signs of GvHD was seen in 2 out of 3 evaluablepatients.

A seventh patient had received a donor liver, but unfortunately haddeveloped sepsis as a result of a surgical complication accompanied byrenal failure due to cyclosporin toxicity and severely depressed bonemarrow function. Because of her condition, the surgeon did not wish torisk the patient or a second liver that had been allocated by usingcurrently available immunosuppressive agents (OKT3, Mofetil, FK506,cyclosporin and ATG) for induction. The patient received the livertransplant and treatment with LO-CD2a for seven days at 5 mg/day withthe first dose infused during surgery beginning prior to declamping ofthe transplanted organ. The subsequent doses were given with low dosesteroids. During the treatment period, the patients renal functionimproved sufficiently for a conventional immunosuppressive regimen to beinitiated. The patient has not shown any signs of rejections at eightweeks post transplant.

Although the present invention, in a preferred embodiment, is directedto inhibition of graft rejection, it is to be understood that the scopeof the invention is not limited thereto and is generally useful for theinhibition of T-cell activation for any and all purposes.

EXAMPLE 6 Construction and Expression of Chimeric Antibody A. Cloningand Sequencing of V_(H) and V_(L) of LO-CD2a

Total RNA was isolated from the cell line LO-CD2a (ATCC HB 11423)according to the method of Chirgwin (Biochemistry, 18:5294, 1979). mRNAwas then prepared using The oligotex-dT mRNA kit (Qiagen, Chatsworth,Calif.). Approximately 200-300 ng mRNA was reverse transcribed using theRNA-PCR kit from Perkin-Elmer Cetus (Norwalk, Conn.). The reaction wascarried out at 42_C for 1 hour. Oligonucleotide primers required foramplification of V_(H) and V_(L) genes were chosen using the followingreferences: 1) Sequences of Proteins of Immunological Interest, Kabat etal., 5th ed., 1991, 2) Orlandi et al., Proc. Nat'l. Acad. Sci., (USA)86:3833-3837 (1989).

V_(L) sense (SEQ ID NO:1)        Sma 1   #1  2   3   4  5   6   7   85′                                         3′    AA CCC GGG GAC ATT CAGCTG ACC CAG TCT CAA C_(L) antisense (SEQ ID NO:2)       Sal 1  #115 114113 112 111 110 109 5′                                       3′    CAGTC GAC TAC AGT TGG TGC AGC ATC AGC V_(E) sense (SEQ ID NO:3)      Sma 1    #1  2  3   4   5   6  7   85′                                        3′    AA CCC GGG GAG GTC CAGCTG CAG CAG TCT GG CH₁ antisense (SEQ ID NO:4)      Sal 1    #124 123122 121 120 119 5′                                    3′    AAG TCG ACCCAG TGG ATA GAC CGA TGG

The numbers refer to amino acid residues, as shown in Kabat, et al.,1991.

Polymerase chain reactions (PCR) were carried out in a Perkin-Elmer DNAThermal Cycler 480 using the following conditions: 5 minutes at 94° C.,30 cycles consisting of 1 minute at 94° C., 2 minutes at 60° C., and 2minutes at 72° C. This was followed by 5 minutes at 72° C. DNA fragmentswere gel purified from 1% agarose using the Qiaex gel extraction kit(Qiagen, Chatsworth, Calif.). The fragments were then blunt-endedaccording to the method of Kanungo and Pandey, BioTechniques, 14:912-913(1993) and ligated into the Sma I site of Bluescript KSII⁺ (Stratagene,La Jolla, Calif.). Multiple clones were sequenced by the dideoxy chaintermination method using the Sequenase ™ T7 Polymerase Kit (U.S.Biochemical, Cleveland, Ohio).

Due to the potential error rate inherent in PCR,at least three separatereactions were performed. The most commonly observed sequences forLO-CD2a V_(L) and V_(H) genes are shown in FIGS. 29 and 30, wherein FIG.29 shows the nucleotide and amino acid sequences of the LO-CD2a V_(L)chain including the native leader sequence. FIG. 30 shows the nucleotideand amino acid sequences of the LO-CD2a V_(H) chain including the nativeleader sequence.

As shown in FIG. 29, the leader sequence is from amino acid residues −20to −1. Framework 1 is from amino acid residues 1 to 23. CDR1 is fromamino acid residues 24 to 39. Framework 2 is from amino acid residues 40to 54. CDR2 is from amino acid residues 55 to 61. Framework 3 is fromamino acid residues 62 to 93. CDR3 is from amino acid residues 94 to102. Framework 4 is from amino acid residues 103 to 112.

As shown in FIG. 30, the leader sequence is from amino acid residues −19to −1. Framework 1 is from amino acid residues 1 to 30. CDR 1 is fromamino acid residues 31 to 35. Framework 2 is from amino acid residues 36to 49. CDR2 is from amino acid residues 50 to 66. Framework 3 is fromamino acid residues 67 to 98. CDR3 is from amino acid residues 99 to107. Framework 4 is from amino acid residues 108 to 118.

B. Insertion into Vectors for Transient Expression

Two vectors were licensed from The Medical Research Council (MRC) inLondon for expression of chimeric light and heavy chains of LO-CD2arespectively. The 9.2 Kb light chain vector (hcmv-vllys-kr-neo) containsthe genomic clone of the human kappa constant region and humanized V_(L)domain of anti-lysozyme as a Hind III-Bam HI fragment. The 8.6 kb heavychain vector (hcmv-VhLys-gamma1-neo) contains the genomic clone of human1 constant region and the humanized V, domain of anti-lysozyme as a HindIII-Bam HI fragment. These vectors are more fully described in Maeda, etal., Hum. Antibod. Hybridomas2:124-134, (1991).

Since DNA fragments containing the native signal peptides wereunavailable, the V regions of LO-CD2a were cloned behind the signalsalready present in the MRC vectors. The light chain V region with signalfragment was constructed from two fragments, each derived from aseparate PCR reaction as follows:

Reaction 1: The DNA template was the MRC light chain vector. Thefragment amplified contained the signal peptide plus a portion offramework (FR)1. The two oligonucleotides used were:

                              Hind III 5 VLlyssig (sense):5′ CCGCAAGCTTCATGGGATGGAG 3′ (SEQ ID NO:5)                                   TthIII 3 VLlyssig (antisense):5′ GCTGCTTGGGGACTGGTCAGCTGGAT 3′ (SEQ ID NO:6)

The antisense primer contained the FR 1 sequence of LO-Cd2a, not thatfound in the MRC vector for anti-lysozyme. The PCR reaction produced a0.15 Kb Hind III—Tth III fragment.

Reaction 2: The DNA template was the LO-CD2a V_(L) clone in Bluescript.The fragment amplified included LO-CD2a FR 1 (from the Tth III site) tothe end of FR 4. The 3′ untranslated region found in the MRC light chainvector was added to the 3′ end of LO-CD2a using the antisenseoligonucleotide. The 2 oligonucleotides used were: 5′ V_(L) LO-CD2a(sense):

            Tth III 5′ ATTCAGCTGACCCAGTCTCCA 3′ (SEQ ID NO:7)3 VL Lo-CD2a (antisense):        BamHI5′ GATCGGATCCACCTGAGGAAGCAAAGTTTAAATTCTACTCACGTTTCAGTTCCAGCTT 3′ (SEQ IDNO:8)

This reaction yielded a 0.35 Kb TthIII-Bam HI fragment. Both PCRproducts were gel purified using Qiaex and restricted with theappropriate enzymes. The Hind III—Tth III fragment plus the Tth III—BamHI fragment then were ligated between the Hind III and Bam HI sites ofBluescript in a 3-way ligation. This construct, containing the entireV_(L) region of LO-CD2a plus the MRC signal peptide was then sequenced.

The heavy chain LO-CD2a V region construct contains the MRC signalsequence at its 5′ end and the long 3′ untranslated region, also derivedfrom the MRC H chain vector. The final construct was made from 3separate PCR reactions as follows:

Reaction 1: The DNA template was the MRC H chain vector. Since V_(L) andV_(H) genes of anti-lysozyme use the same signal, the sense primer wasthe same as that used for the LO-CD2a V_(L) construct, i.e., 5′ V₁lyssig. The antisense primer was 3′ V_(H)lyssig:

                    Pst 1 5′TCTCCTGCAGTGGGACCTCGGAGTGGACACC3′ (SEQ IDNO:9)

This reaction produced a 0.16 Kb Hind III—Pst I fragment containing theMRC signal plus a portion of FR 1 of LO-CD2a. The fragment was gelpurified, restricted, and ligated into Hind III—Pst I cut Bluescript forsequencing.

Reaction 2: The DNA template was the LO-CD2a V_(H) region in Bluescript.This reaction yielded a 0.3 Kb Pst I—Sty I fragment containing most ofthe V_(H) region. Because there was an internal Pst I site in FR 3 ofLO-CD2a, the Pst I—Sty I fragment had to be constructed from 2 PCRreactions as follows:

                                2                   4                            <--------            <----- Pst1                       Pst 1 Sty 1                0.2 Kb                        0.1 Kb FR1                             FR3 FR4------->                       ------->  1                               3The template DNA shown above is clone 82-8, LO-CD2a V_(H) in Bluescript.Reaction A: Yields a 0.2 Kb fragment, using olignucleotides, alsorefered to as oligos 1 and 2, as primers:

Oligo 1 is: 5′Pst I 82-8 (sense):        Pst I                                5′GAGGTCCAGCTGCAGCAGTCT3′ (SEQ ID NO:10)Oligo 2 is:              3′int.   Pst   I    (antisense):5′CGATGTATCAGCTGTCAGTGTGGC3′ (SEQ ID NO:11)Reaction B: Yields a 0.1 Kb fragment, using oligos 3 and 4 as primers.Oligo 3 is 5′ int. Pst I (sense):

                         5′GCCACACTGACAGCTGATACATCG3′ (SEQ ID NO:12)Oligo 4 is 3′Sty I 82-8 (antisense):                                         Sty I                                5′CAGAGTGCCTTGGCCCCAGTA3′ (SEQ ID NO:13)Oligos 2 and 3 above contain changes in nucleotide sequence which removethe internal Pst I site without changing the amino acid sequence ofLO-CD2a. Aliquots (2-5 μl) of the overlapping products of reactions A &B above were combined and served as templates for a third PCR reaction.The oligonucleotide primers for this reaction were numbers 1 and 4 fromthe previous diagram. The 0.3 Kb product was gel purified by Qiaex andrestricted with Pst I and Sty I. Since the fragment remained intact, theinternal Pst 1 site had been successfully mutated.Reaction 3: The final V_(H) fragment was produced using the MRC heavychain vector as template. This 0.23 Kb Sty I—Bam HI fragment contained aportion of FR4 of Lo-CD2a, and the entire 3′ untranslated region fromthe MRC vector. The primers used were: 5′V_(H)lys Sty I (sense):

                Sty I 5′TACTGGGGCCAAGGCACCCTCGTCACA3′ (SEQ ID NO: 14)                                  Bam HI 3′V_(H)lys Bam HI (antisense):5′GATCGGATCCCTATAAATCTCTGGC3′ (SEQ ID NO: 15)The resulting fragment was gel purified and restricted with Sty I andBam HI. The Pst I—Sty I and Sty I—Bam HI fragments were then ligatedinto Pst I—Bam HI cut Bluescript for sequencing.

All oligonucleotides were synthesized on an Applied Biosystemssynthesizer. All sequencing reactions were carried out using TheSequenase ™ T7 Polymerase Kit (U.S. Biochemical, Cleveland, Ohio). AllPCRs were carried out using the following protocol: 5 min. at 95° C., 35cycles consisting of 1 min. at 94° C., 1 min. at 50° C., 2 min. at 72°C., a final extension of 5 min. at 72° C.

LO-CD2a V_(L) and V_(H) fragments containing the correct sequences wereremoved from Bluescript and cloned between the Hind III and Bam HI sitesof the MRC light and heavy chain vectors, respectively. For the H chain,the 5′ Hind III—Pst I fragment was first joined to the remainder of theconstruct (PstI—Bam HI) in Bluescript; the entire Hind III—Bam HIfragment was then cloned into the MRC vector.

C. N-Terminal Amino Acid Sequencing of V_(H) and V_(L)

N-terminal amino acid sequence analysis was performed by HarvardMicrochemistry Laboratory in Cambridge, Mass. on samples of LO-CD2aheavy and light chains in order to confirm the sequences obtained usingRNA-PCR. The samples were prepared as follows:

−200 μg of LO-CD2a was applied across a 12% SDS polyacrylamide gel runin the presence of B-mercaptoethanol. Following electrophoresis, theprotein was transferred to a PVDF membrane using a Western transferapparatus. The membrane was stained briefly with Ponceau S, destained in1% acetic acid, and the light and heavy chain bands were dried undervacuum and sent for amino acid analyses and N-terminal sequencing.

The amino acid sequence of the first 20 residues of LO-CD2a V_(H) agreedcompletely with the cloned sequence; however, the sequence of V_(L)indicated that residues 2, 3 and 7 in FR1 were different than thoseencoded by the cloned genes. These differences all reside in the PCRprimer used for cloning purposes, based on a best guess sequenceobtained from the previously cited literature.

D. DNA Sequence Confirmation of N-Terminal Amino Acid Sequence and itsCorrection

In order to correct this sequence and simultaneously clone the nativesignal peptides of both V_(L) and V_(H) of LO-CD2a, RACE-PCR wasemployed was employed (Rapid Amplification of cDNA Ends): mRNA fromLO-CD2a cells was reverse transcribed and the resulting cDNA wasG-tailed at its 3′ end using terminal transferase in the presence ofdGTP. The cDNA was then amplified using a specific 3′ oligonucleotideand a 5′ oligonucleotide complementary to the G-tail. To simplifysubcloning, a suitable restriction site was added to the 5′ end of eacholigonucleotide.

The oligonucleotides used for preparation of cDNA were as follows:

                   Bam H1 Not I    Sal I 3′ oligoVk(VKA) TTGGATCCGCGGCCGCGTCGACTACAGTTGGTGCAGCATCAGC (SEQ ID NO: 16)                   Bam H1 Not 1    Sal 1 3′ oligoVh(CHA) ATGGATCCGCGGCCGCGTCGACCCAGTGGATAGACCGATGG (SEQ ID NO: 17) Theoligonucleotides for RACE-PCR were as follows:                           Xho I     5′ Primer (TV1): 5′ CCA TGG CCT CGAGGG CCC CCC CCC CCC CCC C 3′ (SEQ ID NO: 18)                               Stu I  3′ oligo Vh (BHA) 5′ CCT GTT TAGGCC TCT GCT TCA CCC AGT AC 3′ (SEQ ID NO: 19)                                        Sph I  3′ oligo Vk (BKA) 5′ GGATAA TGG GTA AAT TGC ATG CAG TAA TA 3′ (SEQ ID NO: 20)

RACE-PCR reactions were carried out using the following protocol: 5 min.at 94° C., 40 cycles of 30 sec. at 94° C., 30 sec. at 50° C., and 50sec. at 72° C., followed by a 5 min. extension at 72° C.

PCR products obtained for LO-CD2a V_(L) and V_(H) were gel extractedusing Qiaex. The V_(H) fragment was restricted with Xho I and Stu I andligated into Xho I—Sma I cut Bluescript. The V_(L) fragment wasblunt-ended and ligated into Sma I cut Bluescript. A number of cloneswere sequenced for both light and heavy chain V regions and the signalsequences were identified.

Since signal sequences found in immunoglobulin genes generally haveintrons, these may be important for expression. Genomic clonescontaining the V_(L) and V_(H) leader sequences were identified as well.Genomic DNA was prepared as follows: 4×10⁷ LO-CD2a cells were spun down,washed in cold PBS, spun down, and washed with PBS again. Cells wereresuspended in 0.4ml digestion buffer (with freshly added proteinase K).This mixture was incubated with shaking at 50° C. for 12-15 hours,extracted with an equal volume of phenol/chloroform/isoamyl alcohol, andspun at 1700×g. The aqueous phase was transferred to a clean tube and ½volume of 7.5 M ammonium acetate and 2 volumes of 95% ethanol wereadded. The DNA was pelleted by spinning 2 minutes, 1700×g. The pelletwas washed with 70% ethanol and air dried. The pellet was resuspended in80 ml TE, pH 8.0.

Using genomic DNA obtained from the cell line LO-CD2a as a template, thefollowing oligonucleotides were designated in order to amplify thegenomic leader sequences of both V_(L) and V_(H) as well as portions ofthe framework regions ending at unique restriction sites (Sph I forV_(L), Pst I for V_(H)).

-   -   LVL #430 TGCAAGCTTCATGATGAGTCCTGTCCAGTC (SEQ ID NO:21) Leader        V_(L) sense/Hind III    -   LVH #429 AGTAAGCTTCATGAAATGCAGGTGGATC (SEQ ID NO:22) Leader        V_(H) sense/Hind III    -   PVHA #428 GGGAGATTGCTGCAGCTGGACTTC (SEQ ID NO:23) V_(H)        antisense/Pst I

PCR reactions were carried out as follows: 100 ng genomic DNA fromLO-CD2a cells, 200 pmol each of oligos LVL and BKA (for V_(L) fragment)or 200 pmol each of LVH and PVHA (for V_(H) fragment), 10 μl 1 mm dNTPs,10 μl 10×Pfu buffer. 1 μl (2.5 units) Pfu DNA polymerase (Stratagene, LaJolla, Calif.) deionized water to 100 μl. Pfu was used because of itsgreater accuracy than Taq polymerase.

The reaction conditions were as follows: 5 min. 94° C., 5 min. 50° C.,35 cycles of 1 min. 94° C., 1 min. 50° C., 1 min. 72° C., followed by 5min. at 72° C. The PCR products were gel purified, restricted andligated into Bluescript for sequencing. Once clones containing thecorrect sequence were identified, Bluescript vectors containing theseclones were cut with Hind III and Sph I (V_(L)) or Hind III and Pst I(V_(H)) and the fragments were gel isolated. The 0.75 Kb Hind III—Sph Ifragment was then ligated into Bluescript containing the originalLO-CD2a V_(L) construct from which the Hind III—Sph I fragment had beenremoved. The new construct contained the native LO-CD2a signal plusintron and a corrected FR1 sequence (in agreement with the N-terminalsequence). The 0.16 Kb Hind III—Pst I fragment was ligated intoBluescript containing the original LO-CD2a V_(H) construct from whichthe Hind III—Pst I fragment had been removed. The new constructcontained the native signal+intron. The newly constructed V_(L) andV_(H) fragments were then removed from Bluescript by digestion with HindIII and Bam HI and cloned into the MRC light and heavy chain vectors,respectively, for expression in COS cells.

E. Transient expressoion in COS cells. COS 7 cells were obtained fromthe ATCC (Accession No. CRL-1651) and were grown in Dulbeccols MinimalEssential Medium (DMEM) with 10% fetal bovine serum (PBS). Optimaltransfection was achieved at approximately 50% confluency of adherentcells. In preparation for transfection, plasmid DNA was added to DMEMcontaining NuSerum and DEAE-Dextran/chloroquine diphosphate. COS cellmedium was removed, the DNA mixture was added and the cells incubatedfor 3 hours at 37° C. This medium then was removed, and 10% DMSO in PBSwas added to the cells for 2 minutes and then removed. DMEM with 10% FBSwas added to the cells. After overnight incubation, the medium wasreplaced and the cells were incubated for 2 days at 37° C. Supernatantswere collected for assay by ELISA for the secretion of chimericantibody.

F. Detection of secreted chimeric antibody by ELISA. Secretion ofchimeric antibody was confirmed by assay of supernatants from thetransfected COS cells in an ELISA designed to detect the presence ofhuman antibody (or a portion thereof). Goat anti-human IgG (H+L) wasdiluted in phosphate buffered saline (PBS) to a concentration of 5 μg/mland bound to the wells of ELISA microtiter plates by overnightincubation at 4° C. Plates were washed 3 times-using an ELISA platewasher.

Remaining free sites were blocked by the addition of 200 μl PBScontaining 1% bovine serum albumin (PBS-BSA) for ½ hr. at roomtemperature. Two-fold dilutions were prepared in PBS-BSA of thesupernatants and of a positive control reference standard (purifiedhuman IgG1k). Media alone and/or PBS-BSA alone constituted negativecontrols. Antibody dilutions and controls were added to the wells andincubated at room temperature for 1 ½ hours. Plates were then washed 3times with a plate washer in PBS containing 0.05% Tween20. Theappropriate dilution of a goat anti-human IgG (gamma chainspecific)-horseradish peroxidase (HRP) conjugated antibody or goatanti-human kappa light chain-HRP conjugated antibody was added to eachwell and incubated at room temperature for 1 hour. Plates were washedwith PBS-Tween20 as described above, after which the developingsubstrate, (ABTS) containing hydrogen peroxide, was added. Boundantibody was detected by reading absorbance at a wavelength of 405 nm.

G. Binding specificity of secreted chimeric antibody. Bindingspecificity of the chimeric antibody was evaluated by flow cytometricanalysis of antibody binding to the CD2-expressing mutant Jurkat cellline JRT3-T3-5. The binding profile of the chimeric antibody (humanIgG1) was compared with those of the native rat antibody (IgG2b) and theisotype-matched control MABs (human IgGI and rat IgG2b) which exhibitirrelevant (non-CD-2) binding specificities.

Preparation of JRT3-5 (Jurkat) cell line. The Jurkat cell line wasobtained from the ATCC (Accession No. TIB-153) and was propagated inD-MEM containing 10% fetal bovine serum (FBS), 10% amino acid supplement(NCTC), and 6 mM L-glutamine (complete medium). The cells weremaintained at 37° C. with 10% CO₂ and were passaged three times per weekat a ratio of 1:4 (the cell concentration at passage being approximately3×10⁶/ml). Jurkat cells were harvested, centrifuged to remove spentmedium, and washed in DMEM. The cells were then resuspended in phosphatebuffered saline (PBS) with 0.1% sodium azide (NaAz), and an aliquot wasremoved for cell quantification. The number of viable cells wasdetermined by trypan blue exclusion.

Indirect staining of Jurkat cells. Cell surface staining was carried outin a 96 well U-bottom microtiter plate. Approximately 6×10⁵ cells in avolume of 90 μl were distributed into each well of the microtiter plate.Dilutions of the antibodies to be tested were prepared in PBS with 0.1%NaAz and distributed into the appropriate wells in a volume of 10 μl.Cells were incubated with antibody for 15 minutes at room temperature,after which the cells were washed 3 times by adding PBS with 0.1% NaAzto each well and by centrifuging for 2 minutes at 1900 rpm (SorvallRT6000D). Resuspension of cells was accomplished by gently tapping theplates. Ten ul aliquots of the appropriate fluorescein-isothiocyanate(FITC)-conjugated secondary antibody (anti-human Ig or anti-rat Ig) wasadded to the appropriate wells and incubated at room temperature for 15minutes in the dark. Plates were washed 3 times in PBS with 0.1% NaAz asdescribed above. Stained cells were fixed by the addition of 200 μl of0.5% paraformaldehyde in PBS and were stored at 4° C. (up to 1 week).

Flow cytometric analysis of stained Jurkat cells. Stained cells weretransferred to 12×17 mm polystyrene tubes for acquisition of data usinga Becton-Dickinson FACScan. Data acquisition and analysis were carriedout using LYSIS-II software. CD2-expressing Jurkat cells were incubatedwith the LO CD2a (rat IgG2b)MAB, the chimeric version of LO-CD2a (humanIgG1), and the corresponding isotype matched controls. Bound antibodywas detected using the appropriate FITC-conjugated secondary antibodyaccording to the protocol described above. Analysis shows similarbinding patterns of the native rat LO CD2a and the chimeric human-ratLO-CD2a.

H. Stable Expression in NSO Cells

In order to express the chimeric antibody in a stable transfectant, theglutamine synthetase gene amplification system was obtained fromCelltech Limited (Berkshire, UK). This system is described inBebbington, et al., Biotechnology, Vol. 10, pgs. 169-175 (1992). Theexpression vectors used were pEE6hCMV-B and pEE12. Such vectors aredescribed in published PCT Application Nos. WO86/05807, WO87/04462,WO89/01036, and WO89/10404. Since neither of these vectors contains Ckappa or C gamma 1, genomic clones for these genes were obtained fromthe MRC light and heavy chain vectors, respectively. Both constantregion clones were sequenced in order to obtain restriction maps. Twoconstructs were made in pEE12: the first contained the light chain (V+C)5′ to the heavy chain (V+C); the second construct contained the heavychain 5′ to the light chain.

The strategy involving the light chain was as follows:

1. pEE6hCMV-B and pEE12 each were digested with Xma I and Eco RI.

2. The 5′ 1.93 Kb portion of the chimeric light chain was removed fromthe MRC vector using HindIII and Eco RI. This fragment was used as atemplate for PCR mutagenesis as follows:

PCR oligos LC 5′ Xma 1: 5′-GATCCCCGGGCCACCATGATGAGTCCTGTCCAG-3′ (SEQ IDNO: 24) LC 3′ Msc 1:5′-AGAATGGCCACGTCATCCGACCCCCTCAGAGTTTACTATTCTACTATCCAACTGAGGAAGC-3′ (SEQID NO: 25)The restriction sites are underlined.

The PCR was performed in order to change the 5′ restriction site fromHindIII to Xma I and to add a Kozak consensus sequence at the 5′ end ofthe construct. This is essential for efficient translation (Kozak, M. J.Cell. Biol. 108: 229, 1989). The 3′ PCR oligo is used to remove internalBam HI and Eco RI sites which would interfere with subsequent cloningsteps. The final product of the PCR mutagenesis is an 0.85 Kb Xma I-MacI fragment. PCRs were carried out following instructions supplied withthe TA cloning kit (Invitrogen, San Diego, Calif.). The followingconditions were used for PCR: 2 min. at 94° C. followed by 30 cycles of1 min. at 94° C., 2 min. at 55° C. and 2 min. at 72° C. This wasfollowed by a 5 min. extension at 72° C. Ligations and transformationswere carried out according to kit instructions. A number of clones weresequenced by the dideoxy chain termination method, as describedpreviously. A correct clone was removed from the TA cloning vector bydigestion with Xma I and Msc I. This fragment was gel purified usingQiaex.

3. The 2.7 Kb C kappa fragment was removed from the MRC vector bydigestion with Msc I and Eco RI. The fragment was gel purified usingQiaex.

Using two separate 3-way ligations, the entire chimeric light chain,i.e., 0.85 Kb Xma I/Msc I fragment+2.7 Kb Msc I/Eco RI fragment wasligated into both pEE6hCMV-B and pEE12, each of which were cut with XmaI/Eco RI.

The strategy involving the heavy chain was as follows:

1. Both pEE6hCMV-B and pEE12 were transfected into the E. coli strainDM1. Both vectors were digested with Eco RI and BclI (BclI will only cutif plasmids are propagated in methylase minus bacteria).

2. The chimeric heavy chain was removed from the MRC vector by digestionwith HindIII and Eco RI. The resultant 2.7 Kb fragment was gel purifiedusing Qiaex. This fragment was digested with Nhe I and Bgl II. Thisproduces a 0.7 Kb HindIII/Nhe I fragment and a 2 Kb Nhe I/Bgl IIfragment. Both fragments were gel purified. The 0.7 Kb fragment was thenused as a template for PCR mutagenesis.

PCR oligos HC 5′ Eco RI: (SEQ ID NO: 26)5′-GATCGAATTCGCCACCATGAAATGCAGGTGGATC-3′ HC 3′ Nhe 1: (SEQ ID NO:27)5′-CCAGAAAGCTAGCTTGCCATCCCTATAAATCTCTGGC-3′The restriction sites are underlined.

This PCR is performed in order to change the 5′ restriction site fromHindIII to Eco RI and to add a Kozak consensus sequence. The 3′ oligo isused to remove an internal Bam HI site which would interfere withsubsequent cloning steps. PCRs, ligations and transformations werecarried out as described previously.

Using two separate 3-way ligations, the entire chimeric heavy chain,i.e. 0.7 Kb Eco RI/Nhe I fragment+2.0 Kb Nhe I/Bgl II fragment wasligated into both pEE6hCMV-B and pEE12, each cut with Eco RI/Bcl I.

(Bcl I and Bgl II are compatible restriction sites.)

Final constructs in pEE12, containing both the chimeric light and heavychains were made as follows:

Light chain 5′ to heavy chain: pEE6hCMV-B, which is carrying thechimeric heavy chain, was digested with Bgl II/Bam HI. The 5.1 Kbfragment containing the heavy chain plus the hCMV promoter, was gelpurified and ligated into the Bam HI site of pEE12 which contains thechimeric light chain. Correct orientation was checked by digestion withSal I/Bam HI. The presence of a 0.28 Kb fragment indicates correctorientation.

Heavy Chain 5′ to light chain: pEE6hCMV-B, which is carrying thechimeric light chain, was digested with Bgl II/Bam HI. The 5.9 Kbfragment which contains the light chain plus the hCMV promoter was gelpurified and ligated into the Bam HI site of pEE12 which contains thechimeric heavy chain. Orientation was checked by digestion with SalI/Bam HI, as indicated above.

NS/O cells (Galfre, et al., Meth. in Enzymol., Vol. 73(B) pgs. 3-46(1981), and deposited with the European Collection of Animal CellCultures as ECACC Catalog No. 85110503. were transfected byelectroporation. Transfected cells were selected by growth inglutamine-free medium. Antibody production and binding activity onCD2-expressing Jurkat cells were confirmed as described above.

Functional analysis of the human-rat chimeric antibody shows that itsfunctional properties are similar to those of the rat LO-CD2a antibody.Both inhibit a primary mixed leukocyte reaction (MLR) when nanogramquantities of antibody up to 120 ng/ml are added to the culture.Furthermore, addition of the chimeric antibody to a primary MLR inducesa state of hyporesponsiveness in the responder population to challengewith the original alloantigen or with a third party alloantigen. Thehyporesponsiveness is alloantigen-specific in that challenge withmitogen or tetanus toxoid elicits a proliferative response.

EXAMPLE 7 Construction and Expression of Humanized Antibody A.Construction of Humanized Light Chain

The framework regions from a human V kappa gene designated as HUM5400(EMBL accession X55400), which shares homology with LO-CD2a, were chosenfor humanization of the light chain V region. Below is a comparisonbetween the frameworks of LO-CD2a and HUM5400:

Framework 1                *     * LO-CD2a: D V V L T Q T P P T L L A TI G Q S V S I S C (SEQ ID NO: 28) HUM5400: - - - M - - S - L S - P V -L - - P A - - - - (SEQ ID NO: 29) Framework 2            **               * * LO-CD2a: W L L Q R T G Q S P Q P L I Y (SEQ ID NO:30) HUM5400: - F Q - - P - - - - R R - - - (SEQ ID NO: 31) Framework 3                                             * LO-CD2a: G V P N R F S GS G S G T D F T L K I S G V E A (SEQ ID NO:32)             E D L G V Y YC HUM5400:    - - - D - - - - - - - - - - - - - - - - R (SEQ ID NO:33)            - - - -          - V - - - - - Framework 4 LO-CD2a: F G A GT K L E L K (SEQ ID NO:34) HUM5400: - - Q - - - - - I - (SEQ ID NO:35)

A comparison of the light chain variable region sequences of the ratLO-CD2a, the homologous human variable region, HUM5400, and humanizedLO-CD2a is shown in FIG. 31. The complete amino acid sequence is givenfor the LO-CD2a variable region and residues are numbered according tothe rat sequence. Residues identical to those of the rat in thecorresonding positions in the humanized and HUM5400 sequences areindicated by horizontal dashed lines, whereas non-identical residues aregiven by letter code. The humanized LO-CD2a light chain variable regionis comprised of the HUM5400 framework regions, the rat LO-CD2a CDR's(underlined), and seven rat LO-CD2a framework residues (designated byan*above the rat sequence) which were selected because such residues maybe relevant for maintaining the binding specificity of LO-CD2a.

As shown in FIG. 31, Framework 1 is from amino acid residues 1 to 23.CDR1 is from amino acid residues 24 to 39. Framework 2 is from aminoacid residues 40 to 54. CDR2 is from amino acid residues 55 to 61.Framework 3 is from amino acid residues 62 to 93. CDR3 is from aminoacid residues 94 to 102. Framework 4 is from amino acid residues 103 to112. The leader sequence is from amino acid residues −20 to −1. (FIG.32). The rat amino acid residues which are retained in the frameworkregions are amino acid residues 9 and 12 in Framework 1; amino acidresidues 41, 42, 50, and 51 in Framework 2; and amino acid residue 82 inFramework 3.

A humanized light chain was constructed which contains the CDRs ofLO-CD2a and the variable region frameworks of HUM5400 except for 7unusual residues (*) which were retained from the frameworks of LO-CD2a.The 5′ region was taken from the chimeric light chain construct. This0.43 Kb Hind III/Hph I fragment contains the native signal plus intronand the sequence encoding the first 3 amino acid residues of framework 1which are identical in the rat and human frameworks. The remainder(i.e., the 3′end) of the construct (0.37 Kb), containing the nucleotidesencoding all but the first three amino acids of the variable region, wassynthesized by PCR from 7 overlapping oligonucleotides, ranging in sizefrom 63-81 nucleotides. These long oligonucleotides served as templatesfor shorter 5′ and 3′ outside PCR oligonucleotides (21-26 nucleotides inlength). In all cases, 5 pmol of template was used along with 100 pmolof each outside PCR oligonucleotide. All PCRs were carried out using Pfupolymerase in order to achieve greater fidelity. The procedure was asfollows: 5 min at 95° C., followed by 25 cycles which included 2 min at94° C., 2 min at 55° C. and 2 min at 72° C. This was followed by anadditional 5 min extension at 72° C. The entire synthesis wasaccomplished in 4 steps. In the first step, the first longoligonucleotide was added on to the 0.43 Kb Hind III-Hph I fragment. Thenext 3 sets of overlapping oligonucleotides were then added sequentiallyusing PCR. After synthesis of the entire 0.8 Kb construct was completed,it was gel purified using Qiaex, restricted with Hind III and Bam HI,gel purified again with Qiaex, and ligated to Hind III/Bam HI cutBluescript KS II. A number of clones were sequenced until a correctversion was obtained. The clone was then removed from Bluescript bydigestion with Hind III and Bam HI. The resultant fragment was gelpurified using Qiaex and ligated into the MRC light chain vector whichhad been cut with Hind III/Bam HI. The nucleotide and amino acidsequences of the humanized LO-CD2a light chain V region are shown inFIG. 32.

The overlapping oligonucleotides used in the synthesis of the humanizedlight chain and a description of their use follows:

Oligonucleotide #1:5′GCAAGAGATGGAAGCTGGTTGTCCCAAGGTTACCAATAATGAAGGTGGACTCTGGG (SEQ ID NO:36)          TCATCACAACATCACCATTGGTTCC3′ Oligonucleotide #2:5′CAACCAGCTTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTACATAGTAGTGGAAA (SEQ ID NO:37)              CACCTATTTAAATTGG3′ Oligonucleotide #3:5′AGATTCCAGTTTGGATACCAAATAAATTAGCGGCTGTGGAGATTGGCCTGGCCTTA (SEQ ID NO:38)          GCAACCAATTTAAATAGGTGTTTCC3′ Oligonucleotide #4:5′TTGGTATCCAAACTGGAATCTGGGGTCCCCGACAGGTTCAGTGGCTCAGGGAGTGG (SEQ ID NO:39)          AACAGATTTCACACTCAAAATCAGT3′ Oligonucleotide #5:5′ATGGGTAAATTGCATGCAGTAATAAACCCCCACATCCTCAGCTTCCACTCCACTGA (SEQ ID NO:40)              TTTTGAGTGTGAAATC3′ Oligonucleotide #6:5′TACTGCATGCAATTTACCCATTATCCGTACACGTTTGGACAAGGGACCAAGCTGGA (SEQ ID NO:41)                   AATCAAA3′ Oligonucleotide #7:5′GATCGGATCCAACTGAGGAAGCAAAGTTTAAATTCTACTCACGTTTGATTTCCAGC (SEQ ID NO:42)                 TTGGTCCCTTG3′

Oligonucleotides 1, 3, 5, and 7 are inverse complementary sequences.

Oligonucleotides 2, 4, and 6 are sense strand sequences.

Oligonucleotide #1 overlaps the 0.43 Kb Hind III/Hph I fragment derivedfrom the chimeric light chain construct. This oligonucleotide was addedto the 0.43 Kb fragment by PCR, using the following PCR oligos:

-   (5′) PCR 1A (sense): 5′GATCAAGCTTCATGATGAGTCCT3′ (SEQ ID NO:43)-   (3′) PCR 1A′ (inverse complement):-   5′GCAAGAGATGGAAGCTGGTTG3′ (SEQ ID NO:44)

In a similar manner, oligonucleotides #2 and #3 were stitched togetherby PCR using the following PCR oligos:

-   5′ PCR 2B (sense): 5′CAACCAGCTTCCATCTCTTGC3′ (SEQ ID NO:45)-   3′ PCR 2B′ (inverse complement):-   5′AGATTCCAGTTTGGATACCAA3′. (SEQ ID NO:46)

After PCR, both products were gel purified using Qiaex, and then joinedtogether using a third PCR. The third PCR required the following oligos:

-   -   (5′) PCR 1A    -   (3′) PCR 2B′.

The resultant fragment was gel purified by Qiaex. Oligonucleotides #4and #5 were then joined together by PCR using the following oligos:

-   (5′) PCR 3C (sense): 5′TTGGTATCCAAACTGGAATCTGGG3′ (SEQ ID NO:47)-   (3′) PCR 3C′ (inverse complement):5′ATGGGTAAATTGCATGCAGTAATA3′ (SEQ    ID NO:48)

The fragment was gel purified and added to the previous construct usingPCR oligos:

-   -   (5′) PCR 1A    -   (3′) PCR 3C′.

The final piece was constructed using oligonucleotides #6 and #7 and PCRoligos:

(5′) PCR 4D (sense): 5′ TACTGCATGCAATTTACCCATTAT 3′ (SEQ ID NO:49) (3′)PCR 4D′ (inverse complement): 5′ GATCGGATCCAACTGAGGAAGCAAAG 3′(SEQ ID NO:50)

After gel purification, this fragment was added to the remainder of thehumanized light chain construct by PCR using oligos:

-   -   (5′) PCR 1A    -   (3′) PCR 4D′.

B. Construction of the Humanized Heavy Chain

The framework regions of the human antibody clone Amu 5-3 (GenBankaccession number U00562) were used for the generation of the humanizedheavy chain. Below is a comparison between the frameworks of LO-CD2a andthose of Amu 5-3:

Framework 1 LO-CD2a: E V Q L Q Q S G P E L Q R P G A S V K L S C K A S(SEQ ID NO: 51)          G Y I F T Amu 5-3: Q - - - V - - - A - V KK - - - - - - V - - - - (SEQ ID NO: 52)     - - T - - Framework 2                               * LO-CD2a: W V K Q R P K Q G L E L V G(SEQ ID NO: 53) Amu 5-3: - - R - A - G - - - - W M - (SEQ ID NO: 54)Framework 3          *     *   *       *                 *   * LO-CD2a:K A T L T A D T S S N T A Y M Q L S S L T S E D T (SEQ ID NO: 55)         A T Y F C A R Amu 5-3: R V - M - R - - - I S - - - - E - - R -R - D - (SEQ ID NO:56)     - V - Y - - - Framework 4 LO-CD2a: W G Q G TL V T V S S (SEQ ID NO: 57) Amu 5-3: - - - - - - - - - - - (SEQ ID NO:58)

FIG. 33 shows the heavy chain variable region sequences of the ratLO-CD2a, the homologous human variable region, Amu5-3, and the humanizedLO-CD2a (humanized Vh). The complete amino acid sequence is given forLO-CD2a and residues are numbered according to the rat sequence.Residues identical to those of the rat in the corresponding positions inthe humanized and Amu5-3 sequences are indicated by horizontal dashedlines whereas non-identical residues are given by letter code. Thehumanized LO-CD2a Vh is comprised of the Amu5-3 framework regions, therat LO-CD2a CDRs, and seven rat LO-CD2a framework residues (designatedby an*above the rat residue) which were selected because they may berelevant for maintaining the binding specificity of LO-CD2a. Thevertical lines in CDR3 of the rat and humanized sequences representspaces which were required to align the three sequences because theAmu5-3 has a longer CDR3 than the rat and humanized regions.

As shown in FIG. 33, Framework 1 is amino acid residues 1 to 30. CDR1 isamino acid residues 31 to 35. Framework 2 is amino acid residues 36 to49. CDR2 is amino acid residues 50 to 66. Framework 3 is amino acidresidues 67 to 98. CDR3 is amino acid residues 99 to 107. Framework 4 isamino acid residues 108 to 118. The leader sequence is amino acidresidues −19 to −1. (FIG. 34).

The rat LO-CD2a amino acid residues which are retained in the frameworkregions are amino acid residue 47 in Framework 2; and amino acidresidues 67, 70, 72, 76, 85 and 87 in Framework 3.

A single humanized heavy chain construct was made. This constructcontains the CDRs of LO-CD2a and the frameworks of Amu 5-3, with theexception of 7 residues (*) retained from LO-CD2a. This construct wasproduced in a manner similar to that of the humanized light chain. Inthis case, there were 12 overlapping template oligonucleotides, rangingin size from 69-88 nucleotides. The 12 outside PCR oligonucleotidesranged in size from 21-26. The synthesis was accomplished in 6 steps,adding on a pair of overlapping template oligonucleotides at each step.The final construct (0.7 Kb) was gel purified using Qiaex, digested withHind III and Bam HI, gel purified again, and ligated into Bluescript forsequencing, as described previously. When a correct clone wasidentified, it was removed from Bluescript by restriction with HindIII/Bam HI, and then ligated into the MRC heavy chain vector which hadbeen digested with the same enzymes. The nucleotide and amino acidsequences of the humanized LO-CD2a heavy chain V region is shown in FIG.34.

The overlapping oligonucleotides used in the synthesis of the humanizedheavy chain and a description of their use follows:

-   Oligonucleotide #1 (sense):-   5′GATCAAGCTTCATGAAATGCAGGTGGATCATCCTCTTCTTGATGGCAGTAGCTACA    GGTAAGGCACTCCCAAGTCCTAAACTTGAGAG3′ (SEQ ID NO:59) (Hind III site    underlined)-   Oligonucleotide #2 (antisense):-   5′CACCTGTGAGTTGACCCCTGTTGAAAGAAATCCAAAGATAGTGTCACTGTCTCCCA    AGTGTATGATCTCTCAAGTTTAGGACTTGGG3′ (SEQ ID NO:60)-   Oligonucleotide #3 (sense):-   5′ACAGGGGTCAACTCACAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCC    TGGGGCCTCAGTGAAGGTCTCC3′ (SEQ ID NO:61)-   Oligonucleotide #4 (antisense):-   5′GGCCTGTCGCACCCAGTACATATAGTACTCGGTGAAGGTGTATCCAGAAGCCTTGC    AGGAGACCTTCACTGAGGCCCC3′ (SEQ ID NO:62)-   Oligonucleotide #5 (sense):-   5′ATGTACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGCTGATGGGAAGGATCGA    TCCTGAAGACGGTAGTATTGAT3′ (SEQ ID NO:63)-   Oligonucleotide #6 (antisense):-   5′TGTGCTAGAGGACGTGTCAGCGGTCAGGGTGACCTTTTTCTTAAACTTCTCAACAT    AATCAATACTACCGTCTTCAGG3′ (SEQ ID NO:64)-   Oligonucleotide #7 (sense):-   5′GCTGACACGTCCTCTAGCACAGCCTACATGGAGCTGAGCAGCCTGACCTCTGACGA    CACGGCCGTGTATTACTGTGCGAGAGGA3′ (SEQ ID NO:65)-   Oligonucleotide #8 (antisense):-   5′GGACTCACCTGAGGAGACGGTGACCAGGGTTCCTTGGCCCCAGTAAGCAAACCTAT    AGTTAAACTTTCCTCTCGCACAGTAATACAC3′ (SEQ ID NO:66)-   Oligonucleotide #9 (sense):-   5′ACCGTCTCCTCAGGTGAGTCCTTACAACCTCTCTCTTCTATTCAGCTTAAATAGAT    TTTACTGCATTTG3′ (SEQ ID NO:67)-   Oligonucleotide #10 (antisense):-   5′CCTAGTCCTTCATGACCTGAAATTCAGATACACACATTTCCCCCCCAACAAATGCA    GTAAAATCTATTT3′ (SEQ ID NO:68)-   Oligonucleotide #11 (sense):-   5′TTCAGGTCATGAAGGACTAGGGACACCTTGGGAGTCAGAAAGGGTCATTGGGAGCC    CGGGCTGATGCAGACA3′ (SEQ ID NO:69)-   Oligonucleotide #12 (antisense):-   5′GATCGGATCCCTATAAATCTCTGGCCATGAAGTCTGGGAGCTGAGGATGTCTGTCT    GCATCAGCCCGGGCTC3′ (SEQ ID NO:70)

overlapping oligonucleotides #1 and #2 were joined together by PCR usingthe following PCR oligos:

-   -   (5′) PCR 4H (sense):

-   5′GATCAAGCTTCATGAAATGCAGGTG3′ (SEQ ID NO:71)    -   (3′) PCR 4H′ (antisense):

-   5′CACCTGTGAGTTGACCCCTGTTG3′.(SEQ ID NO:72)

The resulting fragment was gel purified.

Overlapping oligonucleotides #3 and #4 were joined together by PCR usingthe following PCR oligos:

-   -   (5′) PCR 1E (sense): 5′ACAGGGGTCAACTCACAGGTG3′ (SEQ ID NO:73)    -   (3′) PCR 1E′ (antisense):

-   5′GGCCTGTCGCACCCAGTACAT3′.(SEQ ID NO:74)

The fragment was gel purified.

Oligonucleotides #5 and #6 were joined together by PCR using thefollowing PCR oligos:

-   -   (5′) PCR 2F (sense): 5′ATGTACTGGGTGCGACAGGCC3′ (SEQ ID NO:75)    -   (3′) PCR 2F′ (antisense):

-   5′TGTGCTAGAGGACGTGTCAGC3′.(SEQ ID NO:76)

After gel purification, this fragment was joined to the fragmentproduced by oligonucleotides #3 and #4 by PCR with the following oligos:

-   -   (5′) PCR 1E (sense)    -   (3′) PCR 2F′ (antisense).

The resultant fragment was gel purified.

Oligonucleotides #7 and #8 were joined together by PCR using thefollowing PCR oligos:

-   -   (5′) PCR 3G (sense): 5′GCTGACACGTCCTCTAGCACA3′ (SEQ ID NO:77)    -   (3′) PCR 3G′ (antisense):

-   5′GGACTCACCTGAGGAGACGGT3′. (SEQ ID NO:78)

The resultant fragment was gel purified and added to the construct madeby joining oligonucleotides #3 through #6. This was achieved using PCRoligos.

-   -   (5′) PCR 1B (sense)    -   (3′) PCR 3G′ (antisense).

The resultant fragment, made by joining oligonucleotides #3 through #8,was gel purified. The 5′ end of the construct (oligonucleotides #1+#2)was then added using PCR oligos.

-   -   (5′) PCR 4H (sense)    -   (3′) PCR 3G′.

This fragment was gel purified and the next piece (3′) was added usingoligonucleotides #9 and #10.

Oligonucleotides #9 and #10 were joined by PCR using the following PCRoligos:

-   -   (5′) PCR 5I (sense): 5 ′ACCGTCTCCTCAGGTGAGTCC3′ (SEQ ID NO:79)    -   (3′) PCR 5I′ (antisense):    -   5′CCTAGTCCTTCATGACCTGAA3′.(SEQ ID NO:80)

After gel purification, this 3′ piece was added to the remainder of theconstruct using PCR oligos.

-   -   (5′) PCR 4H (sense)    -   (3′) PCR 5I′ (antisense).

The resultant fragment was gel purified and joined to the remainder ofthe construct.

Oligonucleotides #11 and #12 were joined by PCR using the following PCRoligos:

-   -   (5′) PCR 6J (sense): 5′TTCAGGTCATGAACGACTAGG3′ (SEQ ID NO:81)        and    -   (3′) PCR 6J′ (antisense):    -   5′GATCGGATCCCTATAAATCTCTGGCC3′ (SEQ ID NO:82)

After gel purification, this final 3′ fragment was added to the rest ofthe construct using oligos (5′) PCR4H (sense) and (3′) PCR6J′(antisense). The resultant 0.7 kb final construct was gel purified,sequenced, and cloned into the MRC heavy chain vector, as indicatedpreviously.

Transient expression in COS cells and detection of secreted antibodywere carried out as described previously for the chimeric antibody. Thehumanized antibody was purified by affinity chromatography (Protein A).Binding studies on Jurkat cells demonstrate similar binding patternsbetween the humanized and rat forms of LO-CD2a (FIG. 35). The Jurkatcell line, which expresses CD2, was stained with rat LO-CD2a, thehumanized LO-CD2a (LO-CD2aHu), or rat IgG2b or human IgG controls.Antibody concentrations ranged from 0 to 4 μg/ml. The rat and humanizedforms of LO-CD2a bind to Jurkat cells with similar binding partterns,whereas the rat and human isotype control antibodies do not. Boundantibodies were detected with an isotype-specific FITC-conjugatedantisera. The results shown in FIG. 35 are expressed as the percentageof cells positively stained by the antibodies over the range ofconcentrations mentioned hereinabove. Functional studies indicate thatthe humanized antibody is also capable of inhibiting a primary MLR.Nanogram quantities of LO-CD2a, LO-CD2aHu, rat IgG2b control, or humanIgG1 control were added to culture wells containing equivalent numbersof human peripheral blood mononuclear cells (PBMC) from a designatedresponder and stimulator (irradiated cells). Control wells contained noantibody. The cultures were incubated for 5 days, then pulsed overnightwith tritiated thymidine. (³HT). Proliferation is detected by the uptakeof ³HT. The results, as given in Table 2 below, are expressed as themean CPM as recorded by a Beta plate reader.

TABLE 2 Inhibition of Primary Allogeneic MLR by LO-CD2a and LO-CD2aHuAdditions to Culture Mean CPM No antibody 70,636 LO-CD2a 32,519LO-CD2aHu 27,385 Rat IgG2b 90,859 Human IgG1 88,267 Autologous cellsonly 1,759 Stimulator cells only 115

The humanized antibody also induces a hyporesponsive state to challengewith alloantigen in T cells (as measured by uptake of ³HT as mean CPM)when those T cells are cultured in a primary MLR in the presence ofalloantigen and humanized LO-CD2a but not when an isotype control (withirrelevant specificity) is substituted for the humanized antibody (FIG.36). The functional properties of the humanized antibody are similar tothose of the rat LO-CD2a.

EXAMPLE 8 LO-CD2a Elicits Alloantigen Specific Hyporesponsiveness

The ability of T cells to recognize alloantigen in a secondary MLRfollowing LO-CD2a addition only to the primary culture was examined.Primary MLR cultures contained responder cells and irradiated stimulatorcells in the presence of either LO-CD2a, control antibody (LO-DNP11,Biotranplant, Inc., Charlestown, Mass.), or no antibody, and wereincubated for 7 days. Cells were then washed and rested in media alonefor 3 additional days. After the rest period, cultured cells werere-challenged with the original stimulator cells or cells obtained froma different donor (“third party” cells).

Results of a representative experiment from these studies areillustrated in FIG. 37. In Panel A, a primary response was observed whenresponder cells were cultured in the presence of control antibody at 200ng/ml but when cells were cultured in the presence of LO-CD2a at 200ng/ml no response was observed, consistent with previously reporteddata. The kinetics of the response were determined by harvestingcultures at days 3, 5, and 7. Data were presented as mean values oftriplicate wells run at each data point in which the cpm from individualwells were within 10% of the mean.

As depicted in FIG. 37B, responder cells from cultures treated eitherwith LO-CD2a or control antibody were rechallenged with alloantigenbearing stimulator cells at a 1:1 ratio without any antibody present inthe secondary MLR. The kinetics of the response were determined byharvesting cultures at days 3, 5, and 7. The response of the cellscultured in primary MLR with either LO-CD2a or control antibody isincluded as a control. Data are presented as mean values of triplicatewells run at each data point in which the cpm from individual wells waswithin 10% of the mean. The data are from the same experiment depictedin FIG. 37A.

As shown in FIG. 37C, cells stimulated in a primary MLR with a specificalloantigen in the presence of LO-CD2a or control antibody then werechallenged with alloantigen bearing cells from a third-party donor at a1:1 ratio without any antibody present in the secondary culture. Thekinetics of the response were determined by harvesting cultures at days3, 5, and 7. The response of cells to autologous stimulator cellscultured in the primary MLR with control antibody or LO-CD2a is includedas a control. The data are presented as mean values of triplicate wellsrun at each data point in which the cpm from individual wells werewithin 10% of the mean. The data are from the same experiment asdepicted in FIGS. 37A and B.

Cells cultured in the presence of control antibody in the primaryculture were responsive to re-challenge by the same allogeneicstimulator cells as those in the primary culture (Panel B) and were alsoresponsive to stimulation by third-party cells (Panel C). In contrast,cells cultured in the presence of LO-CD2a during the primary MLR werenot responsive either to rechallenge with the primary allogeneicstimulator cells (Panel B) or stimulation by third-party cells (PanelC). The cells in the cultures containing LO-CD2a were viable andresponsive to stimuli other than alloantigen. For example, stimulationby either PHA or soluble OKT3 evoked equivalent proliferative responsesin cells cultured with LO-CD2a control antibody, or with freshautologous PBMC (data not shown). Flow cytometric analysis of thesecells after rest failed to detect LO-CD2a on the cell surface (data notshown). Thus, these data indicate that exposure to LO-CD2a andalloantigen can induce a state of subsequent alloantigenhyporesponsiveness, i.e., tolerance.

To address the apparent alloantigen specificity of thehyporesponsiveness induced by LO-CD2a during alloantigen stimulation,cells obtained from cultures after alloantigen stimulation werechallenged to respond to the soluble protein antigen, tetanus toxoid.Soluble antigen responses require the presence of viable antigenpresenting cells (APC), which are depleted in alloantigen stimulatedcultures after 7 days. Therefore, in these studies, a fresh source ofAPC was provided to the cultured cells by adding irradiated autologousPBMC to the secondary assay culture. As depicted in FIG. 38A, respondercells from cultures treated with either LO-CD2a or control antibody wererechallenged with alloantigen bearing stimulator cells at a 1:1 ratiowithout any antibody present in the secondary MLR. The kinetics of theresponse were determined by harvesting cultures at days 3, 5, and 7. Theresponse of cells to autologous stimulator cells cultured in primary MLRwith either LO- CD2a or control antibody also is included as a control.The data are presented as mean values of triplicate wells run at eachdata point in which cpm from individual wells were within 10% of themean.

As depicted in FIG. 38B, responder cells from cultures treated witheither LO-CD2a or control antibody were rechallenged with 7.5 μg/mltetanus toxoid without any antibody present in secondary cultures. Thekinetics of the response were determined by harvesting cultures at days3, 5, and 7. The response of cells cultured in primary cultures with noantibody and autologous stimulator cells served as a control forresponse to tetanus toxoid.

The results shown in FIGS. 38A and B demonstrate that although cellscultured with LO-CD2a plus alloantigen in a primary MLR werehyposensitive to alloantigen in a secondary MLR, the cells wereresponsive to tetanus toxoid when presented by fresh APC.

EXAMPLE 9

To address the role of the Fc portion of LO-CD2a, studies were performedto compare the functional effects of the F(ab′)₂ fragment with the wholeantibody.

As shown in FIG. 39, PBMC were cultured for 6 days with irradiatedEpstein-Barr Virus (EBV) transformed B cell line at a 2:1 responder tostimulator ratio and increasing doses of LO-CD2a or its F(ab′)₂fragment. The graph depicts the effect of F(ab′)₂ fragment on theresponse of four different donors, and each point is a mean value oftriplicate wells run at each data point. The results are reported as apercent of control response. For clarity on the graph, the data from theaddition of the whole LO-CD2a antibody are not displayed. At a dose of30 ng/ml of whole LO-CD2a, the responses of the donors were: donor1-18%; donor 2-6.6%; donor 3-3.7%; and donor 4-12% of control. Thecellular responses without antibody present from the individuals testedhad mean values which ranged from 56,690 to 404,843 cpm, and the cpmfrom individual wells were within 10% of the mean.

In order to evaluate further the potential inhibitory properties of theF(ab′)₂ fragment, a dose titration of the F(ab′)₂ fragment was added tounfractionated PBMC stimulated with soluble OKT3 (an APC-dependentresponse). As depicted in FIG. 40, PBMC were stimulated with solubleOKT3 (100 ng/ml) for 3 days. Either intact antibody or the F(ab′)₂fragment of LO-CD2a was added at day 0 at increasing doses. The resultsare expressed as percent of the proliferative response of the cells toOKT3 in the presence of an isotype matched control monoclonal antibody.Each data point is a mean of triplicate wells in which the cpm fromindividual wells were within 10% of the mean. The mean cpm forstimulated PBMC without antibody was 60,117.

The results in FIG. 40 show that T-cell proliferation to OKT3 was notinhibited when the F(ab′)² fragment was used.

EXAMPLE 10 APC are Required for the Inhibitory Properties of LO-CD2a inin vitro Cultures

To address the question of the role of APC in the inhibitory propertiesof LO-CD2a, the T cell population of the PBMC was depleted of CD14,CD56, CD19 and HLA-DR positive cells by immunomagnetic selection.Analysis by flow cytometric techniques of the purified cellsdemonstrated that the APC population was depleted by >95% (data notshown). Proliferation of these purified T cells to soluble OKT3 (an APCdependent response) was reduced to <1% of the proliferation ofunfractionated PBMC by this depletion (data not shown). Purified T cellsor unfractionated PBMC were stimulated by plate bound OKT3 (an APCindependent method of T cell stimulation) in the presence of absence ofLO-CD2a. The ability of LO-CD2a to inhibit T cell activation waseliminated when APC were removed (FIG. 41). As shown in FIG. 41, PBMC orPBMC depleted of APC by immunomagnetic techniques were plated in 96 wellplates coated with OKT3 (10 μg/ml) and cultured for 3 days. LO-CD2a wasadded at the initiation of the cultures. The results are expressed as apercent of the proliferative response of the cells to OKT3 in thepresence of an isotype matched control monoclonal antibody. The plotteddata are representative of three experiments, wherein each data point isthe mean of triplicate wells in which the cpm from individual wells werewithin 10% of the mean.

EXAMPLE 11 Construction and Analysis of Second Generation HumanizedLO-CD2a (MEDI-507) Construction of Vectors

Additional mutagenesis of the first generation of humanized LO-CD2a(Example 7B) heavy chain was performed in order to improve the bindingproperties of the antibody. Four additional rat framework residues wereretianed in addition to the seven retained in the molecule described inExample 7. These four amino acid changes were made using three separatePCR mutagenesis experiments (one of the PCRs led to two amino acidsbeing changed).

All PCRs were carried out using Pfu DNA polymerase (Stratagene, LaJolla, Calif.) and the following program: 5 minutes at 95° C.; 5 minutesat 72° C.; 25 cycles of 1 minute at 94° C.; 1 minute at 55° C., and 1minute at 72° C.; followed by a 5 minute extension at 72° C. Thetemplate DNA was the 701 bp HindIII/BamHI DNA fragment isolated from thefirst generation humanized LO-CD2aV_(H) region. The oligonucleotidesused are listed in Table 3 below.

TABLE 3 Oligonucleotides Used In The Construction of The Heavy Chain OfThe Second Generation Humanized LO-CD2a (MEDI-507) 2nd gen huLO- CD2a-Oligo # Sequence Description Nucleotides 877 GATCAAGCTTCATG 5′ fragmentof Includes 1-21 AAATGCAGGTG huLO-CD2a (HindIII site) (SEQ ID NO: 83)1007 5′ CCCAGGCCTCTG 3′ oligonucleotide reverse CACCTCAGC for changingFR1 complement of (SEQ ID NO: 84) residues (AAs 169-189 12,13) to Gln,Arg 1006 5′ GCTGAGGTGCAG 5′ PCR 169-192 AGGCCTGGGGCC oligonucleotide(SEQ ID NO: 85) for mutating huLO- CD2a VH(AAs 12,13) to Gln, Arg in FR1875 GATCGGATCCCTAT reverse AAATCTCTGGCCA complement of (SEQ ID NO: 86)679-701 (BamHI site) 1012 5′ GATCCTTCCCAC 3′ oligonucleotide reverseCAGCTCAAGCC for changing FR2 complement of (SEQ ID NO: 87) (AA 48) toVal 275-297 1011 5′ GGCTTGAGCTGG 5 oligonucleotide 275-297 GAAGGATC forchanging FR2 (SEQ ID NO: 88) (AA 48) to Val 1020 5′ GTACTCGGTGAA 3′oligonucleotide reverse GATGTATCCAGA for changing FR1 complement of (SEQID NO: 89) residue (AA 28) to 217-237 Ile 1008 5′ TCTGGATACATC 5oligonucleotide 217-237 TTCACCGAG for changing FR1 (SEQ ID NO: 90)(AA28) residue to Ile

Reaction #1 included oligonucleotides 877 and 1007 and generated a 185bp DNA fragment which retained two additional rat LO-CD2a antibodyresidues in framework FR1 at amino acids (AA) 12 and 13 (Gln 12 and Arg13). (Residues are numbered according to Kabat, et al., Sequences ofProteins of Immunological Interest, Fourth Edition, 1987, U.S.Department of Health and Human Services, Public Health Service, NationalInstitutes of Health.)

Reaction #2 included oligonucleotides 1006 and 875 and generated a 515bp DNA fragment also retaining the two rat residues at AAs Gln12 andArg13.

Reaction #3 included oligonucleotides 877 and 1012 and generated a 290bp DNA fragment retaining the rat LO-CD2a residue at position 48.

Reaction #4 included oligonucleotides 1011 and 875 and generated afragment which also retained the rat residue at position 48.

All of the reaction products were purified using Qiaex (Qiagen,Chatsworth Calif.). The reaction products were then subjected toadditional PCR in order to generate the 701 bp V_(H) fragment, asfollows. Reaction #5 included the reaction products from reactions #1and #2, and oligonucleotides 877 and 875; reaction 6 included thereaction products from reactions #3 and #4, and oligonucleotides 877 and875.

In order to obtain the mutation at position 28, (Isoleucine) PCRs wereset up using template from reaction #5 (i.e., containing Gln12 Arg13).Reaction #7 included oligonucleotides 877 and 1020 and generated a 230bp DNA fragment which retained the rat residue at position 28. Reaction#8 included oligonucleotides 1008 and 875 and generated a 470 bp DNAfragment also retaining the rat residue at position 28. Followingpurification of the reaction products using Qiaex (Qiagen, ChatsworthCalif.). The reaction products were then subjected to additional PCR inorder to generate the 701 bp V_(H) fragment and cloned intopBluescript/Sma (Stratagene, La Jolla, Calif.) for subsequent DNAsequencing.

Following sequence analysis, two fragments were gel isolated using Qiaex(Qiagen, Chatsworth, Calif.): (1) 270 bp HindIII/EcoNI (Hu Vh mut 1+2A)containing 3 of the 4 new amino acid changes; and (2) 430 bp EcoNI/BamHI(Hu Vh mut 3D) containing the fourth amino acid change. The fragmentsthen were ligated into the MRC Heavy chain vector, previously describedin Example 6B, which already contained the human heavy chain constantregion, between the HindIII and BamHI sites, to yield the final clone,Clone #257-20. The light chain expression vector was the same as used inthe first generation expression vector (202-2).

FIG. 42 shows the amino acid sequence of the second generation humanizedLO-CD2a VH fragment (MEDI-507) aligned with the sequences of the ratantibody and the human heavy chain variable sequence used in themodeling process.

Transient Expression in COS Cells Using MRC Vectors

Two vectors were licensed from the Medical Research Council (MRC,London, UK) for expression of the humanized light and heavy chains,respectively (FIG. 43). The 9.2 Kb light chain vector(hcmv-vllys-kr-neo) contains a genomic clone of the human kappa constantregion and the humanized VL domain of antilysozyme as a HindIII-BamHIfragment. The 8.6 Kb heavy chain vector (hcmv-VhLys-gamma1-neo) containsa genomic clone of human γ1 constant region and the humanized V_(H)domain of antilysozyme as a HindIII-BamHI fragment (Maeda, et al., 1991,Hum. Antibod. Hybridomas, Vol. 2, pgs. 124-134). The antilysozyme VL andVH domains were removed by restriction digests, and the humanized V_(L)and V_(H) constructs were inserted in their place. The two plasmids werethen co-transfected into COS-7 cells.

COS-7 cells were obtained from the ATCC (Accession number CRL-1651) andgrown in 10% FBS/DME medium to 40-60% confluence in two 1700 cm² surfacearea corrugated plastic roller bottles (Corning, Corning, N.Y.). Allmedium was removed and replaced with transfection medium [DMEM w/10%NuSerum (Collaborative Research, Inc., Waltham, Mass.),DEAE-Dextran/Chloroquine diphosphate, 0.25 mg heavy chain DNA and 0.25mg light chain DNA for humanized LO-CD2a] at 37° C. The roller bottleswere returned to the incubator and roller bottle apparatus for threehours, after which all medium was removed and replaced with 10% DMSO inPBS at 37° C. After 2 to 5 minutes of contact, the DMSO solution wasremoved and replaced by FBS/DMEM. The roller bottles were returned tothe incubator and roller bottle apparatus for one day at 37° C., afterwhich the medium was removed and discarded (protein accumulation assumedto be too low at this point to harvest), and replaced with freshidentical medium. Following incubation for two or three days at 37° C.,one half of the medium in each roller bottle was removed and replacedwith fresh identical medium. This process was repeated until about threeharvests were obtained (after that point, yield decreased). Thesupernatants collected were assayed for the presence of humanized IgG byELISA.

The resulting antibody was purified from transfected COS cellssupernatants by affinity chromatography (Protein A). The in vitroactivity of this humanized antibody was compared to that of rat LO-CD2ausing 3 experimental systems: binding to Jurkat cells, inhibition of theprimary mixed lymphocyte reaction (MLR), and induction ofhyporesponsiveness to alloantigen in a secondary MLR. The functionalproperties of the humanized antibody produced in the transientexpression system were similar to those of rat LO-CD2a, and were laterconfirmed in stable antibody expression in NSO cells.

Stable Expression in NSO Cells Using Celltech Vector

The bioproduction of recombinant mammalian gene products for clinicaluse generally necessitates the use of higher eukaryotic cells to achieveproper processing of the proteins and full biological activity.Immortalized rodent cells are currently the system of choice for thispurpose due to their ability to grow to high density in inexpensivemedium, their high productivity, and their fidelity ofpost-translational processing. The two preferred systems are: 1)glutamine synthetase (GS) selection in murine myeloma NSO cells(Bebbington, et al., 1992, Bio/Technology, Vol. 10, pgs. 169-175), and2) dihydrofolate reductase (dhfr) amplification in Chinese hamster ovary(CHO-DHFR minus) cells (Kaufman and Sharp, 1982, Journal of MolecularBiology, Vol. 159, pgs. 601-621).

The glutamine synthetase system in NSO cells was utilized for the stableexpression and eventual production of the second generation humanizedLO-CD2a (MEDI-507). NSO cells are glutamine auxotrophs with an absoluterequirement for added glutamine, or the introduction of an exogenous GSgene, for survival (Bebbington, et al., 1992). The expression systemtakes advantage of this property to select for cells carrying the GSgene and immunoglobulin genes in glutamine deficient medium (Cockett, etal., 1990, Biotechnology (N Y), Vol. 8, pgs. 662-667). One method bywhich cells can increase the level of glutamine synthetase is toincrease the gene copy number by DNA amplification, with concomitantamplification of the copy number of nearby genes. Implicit in theincreased gene dosage is an increase in the production of the geneproduct, in this case both glutamine synthetase and immunoglobulin.

The Celltech expression vectors (FIG. 44) used were pEE6hCMV-B and pEE12(described in published PCT Application Nos. WO86/05807, WO87/04462,WO89/01036, and WO89/10404). Since neither of these vectors contained Ckappa or C gamma 1, genomic clones,for these genes were obtained fromthe MRC light and heavy chain vectors, respectively. Both constantregion clones were sequenced in order to obtain restriction maps.

Because of differences between the MRC vectors and the Celltech vectors(the MRC vectors for light and heavy chains are separate; in theCelltech vector, the heavy chain follows the light chain, separated by ahCMV promoter region), it was necessary to make the following changes tothe light and heavy chain constructs.

In order to clone the transiently-expressed humanized BTI-322 lightchain construct into the Celltech vector, the following changes weremade. The light chain clone, Clone #202-2, was digested withHindIII/EcoNI, and the 1.93 Kb fragment was gel purified. This fragmentserved as a template for PCR mutagenesis in order to change the 5′restriction site to XmaI and add a translation initiation consensussequence (Kozak, 1986, Point mutations define a sequence flanking theAUG initiator codon that modulates translation by eukaryotic ribosomes,Cell, Vol. 44, 283-292). This PCR reaction was also used to removeinternal BamHI and EcoRI sites to allow for later cloning steps. Theresultant 0.85 kb PCR product was sequenced and then digested withXmaI/MscI. Clone #202-2 was then digested with MscI/EcoRI, and the 2.7kb fragment was gel isolated. A 3-way ligation of the 0.85 kb and 2.7 kbfragments was performed to insert them into pEE12 between the XmaI andEcoRI sites in the polylinker. The resultant clone was named Clone#237-4.

Insertion of the heavy chain construct into the Celltech vector requiredthe following changes: Clone #257-20 was digested with HindIII/EcoRI andthe entire 2.7 kb heavy chain was gel isolated. This fragment then wasdigested further with NheI/BglII to yield a 0.7 kb HindIII/NheI fragmentand a 2.0 kb NheI/BglII fragment (The BglII site is very close to theoriginal 3′ end of the clone, i.e., 20 bp away from the EcoRI site). The0.7 kb HindIII/NheI fragment then was used as a template for PCRmutagenesis in order to change the 5′ restriction site from HindIII toEcoRI and add a translation initiation consensus sequence (Kozak, 1986,Cell, Vol. 44, pgs. 283-292). The 3′ end also was changed by removing aninternal BamHI site. The resultant fragment was sequenced. When acorrect clone was found, a 3-way ligation was performed as follows: the0.7 kb EcoRI/NheI and 2.0 kb NheI/BglII fragments were ligated intopEE6hCMV-B between the EcoRI and BclI sites in the polylinker (BclI andBglII are compatible sites). After successful bacterial transformationswere identified by PCR, one of these clones, designated Clone #6, wasused to construct the final expression vector.

To do this, the 6 kb BglII/BamHI fragment derived from Clone #6, whichcontained the humanized heavy chain plus its hCMV promoter was insertedinto the BamHI site of the pEE12/humanized light chain vector 237-4.After transformation into E. coli, PCRs were performed using templateDNA obtained from bacterial colonies which had been boiled for 5 minutes(colony PCR) in order to identify clones containing both light and heavychains in the correct orientation. The two oligonucleotide PCR primerswere designed to amplify a region spanning the 3′ end of the light chainto the 5′ end of the heavy chain. Clone #12 was identified as correct.To ensure further that this clone contained the light and heavy chaininserts in the appropriate orientation, plasmid DNA was isolated fromthis clone and the DNA was digested with BamHI/SaII. The release of asmall 0.25 kb fragment from Clone #12 indicated that the 6 kbBglII/BamHI fragment containing the heavy chain and the hCMV promoterwas in the correct 5′ to 3′ orientation (If the orientation werereversed, a BamHI/SalI digest would have yielded a 6.3 kb fragment).

Stable Expression in NSO Cell Lines

Clone #12 DNA, linearized with SalI, was used for transfection of NSOcells according to the Celltech Glutamine Synthetase Gene AmplificationSystem—Manual of Operating Procedures (Version 2: For Use with MyelomaCells, Revision 5:6-14, 11-5-92). Cells were electroporated at 3 μF,1500 volts, 2 pulses (40 μg per 10⁷ NSO cells transfected). Afterelectroporation, cells were plated out on 4-6 plates each of 16,000,4,000, and 1000 cells/well in 50 μL/well of 10% FBS/DME. The followingday, 150 μL/well of glutamine-free IMDM with 10% dialyzed FBS was addedto the plates. Colonies were labeled as they were identified to begrowing on the 96 well plates in glutamine-free medium (suggesting theyhad incorporated the gene for glutamine synthetase that was linked tothe gene for the MEDI-507 antibody gene). From the transfectionsperformed with the DNA mentioned above, a total of 204 colonies werescreened by ELISA and selected by highest titer. Approximately one-thirdof the colonies were chosen for expansion to 48 well plates, incubatedfor five days, and then expanded to T-25 flasks. When significant cellgrowth had occurred in any particular T-25 flask (usually another 3-6days), that flask was split further into two or three T-25 flasks eachat 5×10⁴ viable cells/mL. One of these flasks was used to check antibodyproduction by allowing the flask to grow undisturbed for 2-3 weeks. Theother flasks were used to measure cellular growth rates and to propagatethe various cell lines until it could be determined which cell lineswere worthy of further testing or cloning based on antibody production,cellular growth, and adaptability to serum-free medium growth. Forapproximately five weeks during this time the cells used to propagatethe cell line were simply passaged from flask to flask, not alwaysperforming cell counts before each passage. At the end of this period,subcloning was started on several of the most promising cell lines, asdetermined by the criteria mentioned above. MEDI-507hu2.2.204 was from aplate seeded at 16K cells/well plate. Subcloning was performed on CloneMEDI-507hu2.2.204.x on two plates each at 0.5 and 5 cells/well, andallowed to incubate for approximately three weeks.

The subclone labeled Clone #204.11 was generated on a 5 cells/well plate(initially calculated starting viable cell density). That particularplate had growth in 33 of 96 wells. Twenty-nine clones (from all plates)were labeled and screened by ELISA. Five of the 29 clones were selectedfor expansion to 48 well plates, then to a T-25 flask following 5-7 daysof incubation. All five clones were incubated further for 5-7 days andthen expanded to T-75 flasks. After 5-7 days some of the cells fromthese flasks were used to prepare frozen vials of each clone, the restof the cells were used for further testing of antibody production rate,growth rate, and adaptability to serum-free medium. After the screeningprocess was complete, the previously frozen vials of the best clones ofthe best cell lines were thawed and were expanded to freeze down severalvials (usually 3 or 4) of each, and further evaluated in culture.

Comparisons Of In Vitro Functional And Biological Properties OfHumanized MEDI-507 And Rat LO-CD2a

Studies were conducted comparing the functional and biological activityof the humanized MEDI-507 to that of rat LO-CD2a in terms of relativebinding affinity, inhibition of mixed lymphocyte reaction (MLR), abilityto induce hyporesponsiveness to alloantigen in a secondary MLR, and CD2cell depletion in Hu-CD2 transgenic mice. Results indicate that the twoantibodies behave similarly.

Relative Binding to Jurkat Cells

A Jurkat cell based binding assay was performed to compare thecompetitive binding of a research lot of humanized MEDI-507 (Lot PA112895) with that of rat LO- CD2a (Lot SO82/83). Three hundred AL ofJurkat cells at a cell density of 1.3×10⁶ cells/mL were incubated at 4°C. with 20 μL of a 4 mM solution of ¹²⁵I-labeled rat LO-CD2a and 80 ALof cold (unlabeled) rat LO-CD2a or humanized MEDI-507. The effectiveconcentration of the labeled rat LO-CD2a was 0.2 nM. Unlabeled ratLO-CD2a and humanized MEDI-507 was varied from 2 nM to 0 nM. A tubecontaining 20 nM effective concentration of cold LO-CD2a served as acontrol for non-specific binding.

After 30 minutes of incubation at 4° C., 240 μL of this reaction mixturewas layered on top of a 50 μL silicon oil mixture (2:1 (v/v) of AR-200:Thomas Silicon Oil) in a narrow polypropylene tube and centrifuged at14,000×g for 5 minutes in a microcentrifuge. The separation of thelabeled antibody from the bound antibody was achieved by sedimenting thecells through the oil plug. The oil layer was cut and counted. Onehundred μL of the reaction mixture prior to separation was counteddirectly in order to determine the total cpm input. Analysis of assayresults show similar binding patterns between rat LO-CD2a and humanizedMEDI-507 (FIG. 45).

Inhibition of MLR

The ability of rat LO-CD2a (Lot SO82/83) and two different research lotsof the humanized MEDI-507 (Lots QAS and ASQ) to inhibit a mixedlymphocyte reaction was examined. Irradiated human stimulator PBMC wereincubated with human responder PBMC from a different donor in thepresence of rat LO-CD2a, humanized MEDI-507, and appropriate isotypecontrols at concentrations ranging from 10 to 60 ng/mL. After incubationfor 5 days at 37° C., the cultures were pulsed with ³H-thymidine, andthymidine incorporation was determined. The results (FIG. 46) show thatboth the rat LO-CD2a and humanized MID-507 inhibit MLR similarly, in adose-dependent manner.

Induction of Hyporesponsiveness

An important biological property of rat LO-CD2a is its ability to inducein vitro alloantigen-specific hyporesponsiveness. The ability ofhumanized MEDI-507 (lots QAS and ASQ) to induce hyporesponsiveness wascompared to that of rat LO-CD2a (Lot SO82/83).

Primary MLRs were performed in the presence of 10, 60, and 200 ng/ml andof humanized MEDI-507, rat LO-CD2a, or appropriate isotype controls, anda control culture containing no antibody. After incubation for 7-8 days,cells were isolated from cultures, Ficolled, washed, resuspended inculture medium and allowed to “rest” for 3-4 days at 37° C. Cells fromthese cultures were used as responder cells in a secondary MLR culturecontaining irradiated stimulator cells from the original donor. Thecultures then were incubated for an additional 48 hours, pulsed with³H-thymidine, and thymidine incorporation was determined. Analysis ofresults (FIG. 47) demonstrates that humanized MEDI-507 also induces astate of hyporesponsiveness to challenge with alloantigen.

CD2 Cell Depletion

The effect of humanized MEDI-507 and rat LO-CD2a on human CD2 receptorsgrafted into mice (Hu-CD2 transgenic mice) was compared. Twenty-fiveHu-CD2 transgenic mice were assigned to one of five groups to receivehumanized MEDI-507 (0.12 mg/kg or 0.006 mg/kg), rat LO-CD2a (0.12 mg/kgor 0.006 mg/kg), or a buffer control (5 mice/group). Mice were bled atDays 0 (prior to receiving dose), 1, 3, 7, 14, 21, 28, 36, and 44; bloodwas analyzed for CD2 lymphocytes.

Whole blood was collected into tubes containing heparin. Four 20 μLaliquots were dispensed into 96 well, round-bottomed tissue cultureplates, stained with the appropriate antibodies, and treated with FACSLyse to lyse red blood cells and to fix stained cells. After washing toremove non-bound antibody and cellular debris, the remaining lymphocytepopulation was analyzed by flow cytometry.

FIG. 48 shows that the low dose of the humanized MEDI-507 and ratLO-CD2a (0.006 mg/kg, which is a 0.15 μg dose in these mice) had nodetectable effect on the CD2 cell count; these mice were not testedafter the 14 day time point. The high dose of humanized MEDI-507 and ratLO-CD2a (0.12 mg/kg, which is a 3.0 μg dose in these mice) had a large,depleting effect on the CD2 cell counts. Repletion of CD2-bearing cellsin the transgenic mice treated with high doses of the humanized and ratantibodies also was comparable.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1. A humanized antibody comprising the CDRs from LO-CD2a, Produced bythe cell line deposited as ATCC HB 11423 said humanized antibodycontaining in the framework of the heavy chain variable region of thehumanized antibody, amino acids 47, 67, 70, 72, 76, 85, and 87, and one,two, three, or four of amino acids 12, 13, 28, and 48 of the rat LO-CD2aheavy chain variable region (SEQ ID NO:100) as shown in FIG.
 42. 2. Thehumanized antibody of claim 1 wherein said humanized antibody containsin the framework of the heavy chain variable region of the humanizedantibody, amino acids 12, 13, 28, 47, 48, 67, 70, 72, 76, 85, and 87 ofthe rat LO-CD2a heavy chain variable region (SEQ ID NO:100) as shown inFIG.
 42. 3. The humanized antibody of claim 1 wherein said humanizedantibody further contains in the framework of the light chain variableregion of the humanized antibody, one or more of amino acids 9, 12, 41,42, 50, 51, and 82 of the rat LO-CD2a light chain variable region (SEQID NO:95) as shown in FIG.
 31. 4. The humanized antibody-of claim 3wherein said humanized antibody contains in the framework of the lightchain variable region of the humanized antibody, amino acids 9, 12, 41,42, 50, 51, and 82 of the rat LO-CD2a light chain variable region (SEQID NO:95) as shown in FIG.
 31. 5. The humanized antibody of claim 1wherein the heavy chain variable region of the humanized antibody hasthe amino acid sequence (SEQ ID NO:105) of FIG.
 42. 6. The humanizedantibody of claim 3 wherein the light chain variable region of thehumanized antibody has the amino acid sequence (SEQ ID NO:96) of FIG.31.
 7. A process for inhibiting rejection of a graft in a patient,comprising: treating a patient with the antibody of claim
 1. 8. Aprocess of inhibiting the activation or proliferation of T cells byadministering to a patient the antibody of claim 1.